Large-scale Deletions Research Articles

Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria.

Combining prokaryotic end-joining with targeted endonucleases generates specific mtDNA deletions in human cellsEngineering a panel of cell lines with a large-scale deletion that spans the full spectrum of heteroplasmy75% heteroplasmy is the threshold that triggers mitochondrial and cellular dysfunctionTwo distinct nuclear transcriptional programs in response to mtDNA deletions: threshold-triggered and heteroplasmy-sensing.

Long-term hematopoietic dysfunction in patients with large-scale mitochondrial DNA deletion syndromes.

Pearson syndrome (PS) and Kearns-Sayre syndrome (KSS) are single large-scale mitochondrial DNA deletion (SLSMD) syndromes. PS is characterized by severe, transient childhood cytopenia, whereas KSS typically manifests later in life without hematologic abnormalities. Despite distinct clinical presentations, both share a common mitochondrial DNA deletion. Recent observations suggest a potential link between PS progression and myeloid malignancy development, indicating that bone marrow failure (BMF) may be a key aspect of PS pathology and potentially universal across SLSMDs. This study explores longitudinal hematological manifestations of SLSMD syndromes, focusing on bone marrow (BM) dysfunction. Sixteen patients with SLSMDs (13 PS and 3 KSS) were followed, of whom 75% experienced cytopenia, necessitating blood transfusions in 56%. Despite achieving transfusion independence at a median age of 24 months, persistent hematological abnormalities were noted. Comprehensive longitudinal BM studies were conducted in 62% of subjects and consistently revealed signs of marrow dysfunction, even without concurrent cytopenia. Median BM cellularity at a median age of four years and eight months was 50%, with histological signs of dyserythropoiesis, abnormal megakaryocytes, and signs suggesting myelodysplasia. Reduced CD34+ counts and BM colony-forming unit capacity were noted, alongside chromosome 7 aberrations in 16% of patients on cytogenetic studies. Our findings establish BM dysfunction as a persistent hallmark of SLSMD syndromes, posing a risk of clonal evolution and acquisition of chromosome 7 aberrations. This aligns with recent literature, emphasizing enduring BMF in SLSMD syndromes and advocating for tailored hematological monitoring guidelines for this unique patient cohort.

Large-scale copy number alterations are enriched for synthetic viability in BRCA1/BRCA2 tumors

BackgroundPathogenic BRCA1 or BRCA2 germline mutations contribute to hereditary breast, ovarian, prostate, and pancreatic cancer. Paradoxically, bi-allelic inactivation of BRCA1 or BRCA2 (bBRCA1/2) is embryonically lethal and decreases cellular proliferation. The compensatory mechanisms that facilitate oncogenesis in bBRCA1/2 tumors remain unclear.MethodsWe identified recurrent genetic alterations enriched in human bBRCA1/2 tumors and experimentally validated if these improved proliferation in cellular models. We analyzed mutations and copy number alterations (CNAs) in bBRCA1/2 breast and ovarian cancer from the TCGA and ICGC. We used Fisher’s exact test to identify CNAs enriched in bBRCA1/2 tumors compared to control tumors that lacked evidence of homologous recombination deficiency. Genes located in CNA regions enriched in bBRCA1/2 tumors were further screened by gene expression and their effects on proliferation in genome-wide CRISPR/Cas9 screens. A set of candidate genes was functionally validated with in vitro clonogenic survival and functional assays to validate their influence on proliferation in the setting of bBRCA1/2 mutations.ResultsWe found that bBRCA1/2 tumors harbor recurrent large-scale genomic deletions significantly more frequently than histologically matched controls (n = 238 cytobands in breast and ovarian cancers). Within the deleted regions, we identified 277 BRCA1-related genes and 218 BRCA2-related genes that had reduced expression and increased proliferation in bBRCA1/2 but not in wild-type cells in genome-wide CRISPR screens. In vitro validation of 20 candidate genes with clonogenic proliferation assays validated 9 genes, including RIC8A and ATMIN (ATM-Interacting protein). We identified loss of RIC8A, which occurs frequently in both bBRCA1/2 tumors and is synthetically viable with loss of both BRCA1 and BRCA2. Furthermore, we found that metastatic homologous recombination deficient cancers acquire loss-of-function mutations in RIC8A. Lastly, we identified that RIC8A does not rescue homologous recombination deficiency but may influence mitosis in bBRCA1/2 tumors, potentially leading to increased micronuclei formation.ConclusionsThis study provides a means to solve the tumor suppressor paradox by identifying synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers.

Synthetic lethality and the minimal genome size problem.

Gene knockout studies suggest that ~300 genes in a bacterial genome and ~1,100 genes in a yeast genome cannot be deleted without loss of viability. These single-gene knockout experiments do not account for negative genetic interactions, when two or more genes can each be deleted without effect, but their joint deletion is lethal. Thus, large-scale single-gene deletion studies underestimate the size of a minimal gene set compatible with cell survival. In yeast Saccharomyces cerevisiae, the viability of all possible deletions of gene pairs (2-tuples), and of some deletions of gene triplets (3-tuples), has been experimentally tested. To estimate the size of a yeast minimal genome from that data, we first established that finding the size of a minimal gene set is equivalent to finding the minimum vertex cover in the lethality (hyper)graph, where the vertices are genes and (hyper)edges connect k-tuples of genes whose joint deletion is lethal. Using the Lovász-Johnson-Chvatal greedy approximation algorithm, we computed the minimum vertex cover of the synthetic-lethal 2-tuples graph to be 1,723 genes. We next simulated the genetic interactions in 3-tuples, extrapolating from the existing triplet sample, and again estimated minimum vertex covers. The size of a minimal gene set in yeast rapidly approaches the size of the entire genome even when considering only synthetic lethalities in k-tuples with small k. In contrast, several studies reported successful experimental reductions of yeast and bacterial genomes by simultaneous deletions of hundreds of genes, without eliciting synthetic lethality. We discuss possible reasons for this apparent contradiction.IMPORTANCEHow can we estimate the smallest number of genes sufficient for a unicellular organism to survive on a rich medium? One approach is to remove genes one at a time and count how many of such deletion strains are unable to grow. However, the single-gene knockout data are insufficient, because joint gene deletions may result in negative genetic interactions, also known as synthetic lethality. We used a technique from graph theory to estimate the size of minimal yeast genome from partial data on synthetic lethality. The number of potential synthetic lethal interactions grows very fast when multiple genes are deleted, revealing a paradoxical contrast with the experimental reductions of yeast genome by ~100 genes, and of bacterial genomes by several hundreds of genes.

O-095 Assessment of genome editing outcomes in human preimplantation embryos subjected to CRISPR-Cas9 – most loss of heterozygosity (LOH) events are caused by DNA repair deficiency

Abstract Study question What is the cause of loss of heterozygosity, frequently observed in embryos subjected to CRISPR-Cas9, an interhomolog-homologous recombination repair (IH-HR) or deficiency in DNA repair? Summary answer Vast majority of LOH observed in human zygotes subjected to CRISPR-based editing is explained by deficiency in DNA repair prior to genome activation, not IH-HR. What is known already Genome editing (GE) at the preimplantation stage, intended to correct disease-causing mutations, is controversial due to safety and ethical concerns. It has been suggested that a high frequency of LOH seen in zygotes subjected to GE could be explained by resolution of the double-stranded DNA breaks (DSBs) created using CRISPR-Cas9 by interhomolog-homologous recombination repair (IH-HR), a process with potential to correct deleterious heterozygous mutations. However, such hypotheses are controversial and it is unclear whether embryos at this stage development can utilise IH-HR. CRISPR-Cas9 targeted sites are difficult to interrogate with traditional methods, risking a misinterpretation of editing outcomes. Study design, size, duration We hypothesised that the frequently observed LOH affecting the CRISPR-targeted sites in human zygotes is caused by the deficiency in DNA repair at this developmental stage, resulting in complex genotypes which traditional molecular methods have failed to detect, and not IH-HR which uses the second un-cleaved parental allele as a template for repair. DSBs were induced at three genomic sites in embryos microinjected at the time of fertilisation (n = 27) in this IRB-approved study. Participants/materials, setting, methods Research embryos were cultured for 60 hours, then disaggregated. Individual cells underwent an array of genetic tests, including NGS, allowing determination of the repair pathway utilised and revealing any cytogenetic abnormalities. A novel state-of-the-art analytical workflow was developed to interrogate 25kb around CRISPR cleavage sites. This approach provided a means of detecting complex genomic rearrangements as well as unresolved DNA damage, which would appear as LOH when employing traditional PCR-based approaches for assessment. Main results and the role of chance Induction of DSBs using CRISPR-Cas9 was 100% efficient when applied at the time of fertilisation. LOH was confirmed to be common at targeted sites, affecting 69/216 (32%) loci examined in 108 blastomeres. Remarkably, the great majority of LOH events (>90%) were a direct consequence of inappropriate/failed DNA repair (unresolved DNA breaks or complex genomic rearrangements). Strikingly, not a single instance of IH-HR was observed when CRISPR reagents were introduced at fertilisation. This may be explained by the physical separation of the two parental genomes in distinct pronuclei at that time. Allele-drop out (ADO), a technical problem, accounted for <10% of all LOH events. In total, 46% of DSBs failed to undergo appropriate repair, resulting in segmental aneuploidy (21%), large-scale deletions (10%), inversions/translocations (10%), or loss of the targeted chromosome (5%). 45% of DSBs were resolved using error-prone non-homologous end joining (NHEJ), while only 9% utilised the homology directed repair (HDR) pathway required for correction of mutations. Application of CRISPR-Cas9 to embryos on day-3 of development (after embryonic genome activation - EGA) was less efficient, only producing a DSB in 6/37 embryos (16%). However, the mode of repair differed markedly, 100% employing HDR (p = 0.0001). In 4 cases, this involved IH-HR. Limitations, reasons for caution This data provides strong evidence that LOH recorded in earlier studies was incorrectly attributed to IH-HR and was actually due to abnormal/failed repair. CRISPR appears more effective after EGA, with efficient DNA repair dominated by desirable HDR. However, a larger number of embryos is required to corroborate this finding. Wider implications of the findings The use of CRISPR-Cas9 at the time of fertilisation is frequently associated with problematic, unintended outcomes, rendering the technique clinically inapplicable in the current format. Careful study design which allows determination of all possible editing outcomes is essential and advances our understanding of the underlying mechanisms of DNA repair. Trial registration number not applicable

Mitochondrial defects in sporadic inclusion body myositis-causes and consequences.

Sporadic inclusion body myositis (sIBM) is a distinct subcategory of Idiopathic Inflammatory Myopathies (IIM), characterized by unique pathological features such as muscle inflammation, rimmed vacuoles, and protein aggregation within the myofibers. Although hyperactivation of the immune system is widely believed as the primary cause of IIM, it is debated whether non-immune tissue dysfunction might contribute to the disease's onset as patients with sIBM are refractory to conventional immunosuppressant treatment. Moreover, the findings that mitochondrial dysfunction can elicit non-apoptotic programmed cell death and the subsequent immune response further support this hypothesis. Notably, abnormal mitochondrial structure and activities are more prominent in the muscle of sIBM than in other types of IIM, suggesting the presence of defective mitochondria might represent an overlooked contributor to the disease onset. The large-scale mitochondrial DNA deletion, aberrant protein aggregation, and slowed organelle turnover have provided mechanistic insights into the genesis of impaired mitochondria in sIBM. This article reviews the disease hallmarks of sIBM, the plausible contributors of mitochondrial damage in the sIBM muscle, and the immunological responses associated with mitochondrial perturbations. Additionally, the potential application of mitochondrial-targeted chemicals as a new treatment strategy to sIBM is explored and discussed.

Abstract PO3-24-06: Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers

Abstract Background Pathogenic BRCA1 and BRCA2 (BRCA1/2) germline mutations contribute to hereditary breast cancer and are linked with increased risk of other cancers. Paradoxically, bi-allelic inactivation of BRCA1/2 (bBRCA1/2) is embryonically lethal. Although TP53 loss can at least in part mitigated the deleterious impact of Brca1 deficiency in mice, this is insufficient in human cells to compensate for decreased proliferative capacity. What other compensatory mechanisms facilitate oncogenesis remains unclear. Methods We performed an integrative analysis of cancer genomes from bBRCA1/2 tumors and CRISPR/Cas9 screens from cellular models to identify novel gene candidates that facilitate synthetic viability. First, we analyzed cancer genomes from ovarian and breast cancer derived from TCGA and identified genomic loci, which were enriched for recurrent copy number alterations (CNAs) in bBRCA1/2 tumors. We subsequently identified genes from these CNA loci that had corresponding changes in gene expression. Then, we used high-throughput genome-wide CRISPR/Cas9 knockout screens in BRCA1/2−/− cell lines (BRCA1−/- in RPE1 cells, BRCA2−/− in DLD1 cells) to identify which of these genes increased cell proliferation in vitro. Lastly, we experimentally validated these synthetic viable interactions using an orthogonal MCF12a system to evaluate changes in colony formation with a clonogenic cell survival assay. Results In human cancer genomes, we identified that bBRCA1/2 tumors harbored recurrent large-scale genetic deletions significantly more frequent than histologically matched control tumors. We identified 148 cytobands (23 distinct genomic loci) frequently deleted in bBRCA1/2 ovarian cancers (N=92), and 90 cytobands (15 distinct genomic loci) in bBRCA1/2 ER+ breast cancers (N=30). Subsequent computational analysis of gene expression data identified transcriptionally decreased genes in the frequently deleted loci. Cross-referencing with CRISPR/Cas9 screens in BRCA1/2−/− cells, we ultimately identified 277 and 218 genes that were altered in human tumors and putatively enhanced viability in BRCA1/2-null models but not in isogenic wild-type cell lines in BRCA1 and BRCA2 models, respectively. We subsequently selected 17 genes that either increased viability in BRCA1, BRCA2, or both contexts for experimental validation. Of these, about 40% (7 of 17) were experimentally validated (RIC8A, GNA12, ATMIN, IPO7, ATXN2, KDM1A, and NUP98) and had evidence of synthetic viability in this cellular context. Interestingly, RIC8A and GNA12 interact with each other, are both involved in G protein-coupled signaling pathway, and are synthetically viable with both BRCA1−/− and BRCA2−/− cells. In a re-analysis of a recently published genomic dataset on metastatic breast cancer, we found that homologous recombination deficient tumors developed additional loss of function mutations in RIC8A in the metastatic setting. Conclusions This study provides insights into the oncogenesis of BRCA1/2 malignancies and describes a high-throughput framework to identify synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers. Citation Format: Yingjie Zhu, Xin Pei, Ardijana Novaj, Jeremy Setton, Fatemeh Derakhshan, Pier Selenica, Niamh McDermott, Sonali Sinha, Atif Khan, Simon Powell, Jorge Reis-Filho, Nadeem Riaz. Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-06.

Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus.

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value.IMPORTANCEThere is a critical need to develop novel antibiotics to combat antimicrobial resistance. Streptomyces species are very rich source of antibiotics, typically encoding 20-60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial Streptomyces rimosus strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain S. rimosus ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.

Mitochondrial Deoxyribonucleic Acid Copy Number Elevation As a Predictor for Extended Survival and Favorable Outcomes in High-Grade Brain Tumor Patients: A Malaysian Study.

Investigating the role of mitochondrial DNA (mtDNA) alterations and their impact on brain tumor progression remains a significant focus in cancer research. The research aimed to explore the specific contributions of mtDNA copy number changes and their correlations with patient survival, large mtDNA deletions, and TFAM mutations in brain tumor patients. A total of 41 patients with confirmed brain tumors underwent DNA extraction from both tumor tissues and blood samples. The relative mtDNA copy number in comparison to the nuclear genome was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Long-range PCR assessed largescale mtDNA deletions, and Sanger sequencing was applied to detect exon 4 TFAM mutations. Analysis revealed significantly increased mtDNA copy numbers in brain tumor tissues (80.5%) compared to matched blood samples (P < .001). Median delta Ct (∆Ct) values were 7.35 in cancerous tissues and 11.81 in blood (P <.001), with median relative mtDNA content of 0.0123 and 0.0006, respectively (P <.001). Patients with higher mtDNA copy numbers experienced longer overall survival periods (P=.045) and notably favorable outcomes, particularly in high-grade tumor cases (P=.016). Furthermore, a singlenucleotide deletion was identified in exon 4 of TFAM in a patient with glioblastoma IV, while no large-scale mtDNA deletions were found in brain tumor patients. Our study strongly supports the role of increased mtDNA copy numbers as a reliable predictor for improved survival and positive outcomes in high-grade brain tumor patients.

Cellular and Molecular Responses to Mitochondrial DNA Deletions in Kearns-Sayre Syndrome: Some Underlying Mechanisms.

Kearns-Sayre syndrome (KSS) is a rare multisystem mitochondrial disorder. It is caused by mitochondrial DNA (mtDNA) rearrangements, mostly large-scale deletions of 1.1-10kb. These deletions primarily affect energy supply through impaired oxidative phosphorylation and reduced ATP production. This impairment gives rise to dysfunction of several tissues, in particular those with high energy demand like brain and muscles. Over the past decades, changes in respiratory chain complexes and energy metabolism have been emphasized, whereas little attention has been paid to other reports on ROS overproduction, protein synthesis inhibition, myelin vacuolation, demyelination, autophagy, apoptosis, and involvement of lipid raft and oligodendrocytes in KSS. Therefore, this paper draws attention towards these relatively underemphasized findings that might further clarify the pathologic cascades following deletions in the mtDNA.

Large-Scale Deletions of Mitochondrial DNA in Epilepsy Patients Treated with Carbamazepine

Mitochondrial dysfunction, caused by large-scale deletion mutations, can lead to impaired function of the mitochondrial respiratory chain, reduced ATP production, and serious effects on most energy-consuming organs, such as neurons, and can induce seizures in epilepsy. Carbamazepine (CBZ), the first-line drug used in the treatment of epilepsy, can be harmful to mitochondria and its side effects may be related to mitochondrial dysfunction. In this study, mitochondrial DNA (mtDNA) large-scale deletions were identified in 65 CBZ-treated patients with epilepsy, including 32 patients with CBZ-induced hypersensitivity and 33 with CBZ tolerance. Using the PCR method, mtDNA large-scale deletions were identified in 21/65 epilepsy patients (32.31%), including 9/32 CBZ-hypersensitivity patients (28.13%) and 12/33 CBZ-tolerance patients (36.36%). However, this difference was not statistically significant. The “common deletion” of 4977 bp was the most prevalent deletion. Remarkably, the new deletion of 4876 bp in a CBZ-tolerance patient was reported for the first time. Quantitative PCR analysis showed that the level of mtDNA large-scale deletion was significantly lower in the hypersensitivity group than in the tolerance group (p &lt; 0.05). Besides, analysis of the association between the level of mtDNA large-scale deletion and mtDNA copy number with the clinical features of CBZ hypersensitivity patients showed no relationship with age and severity of skin lesions (p &gt; 0.05). However, there was a statistically significant association between the level of mtDNA large-scale deletions and sex (p &lt; 0.01). Further studies are needed to evaluate the role of mtDNA large-scale deletions in epilepsy and their association with antiepileptic drugs.

Co-expression of prepulse inhibition and Schizophrenia genes in the mouse and human brain

Schizophrenia is a complex psychiatric disorder with genetic and phenotypic heterogeneity. Accumulating rare and genome-wide association study (GWAS) common risk variant information has yet to yield robust mechanistic insight. Leveraging large-scale gene deletion mouse phenomic data thus has potential to functionally interrogate and prioritize human disease genes. To this end, we applied a cross-species network-based approach to parse an extensive mouse gene set (188 genes) associated with disrupted prepulse inhibition (PPI), a Schizophrenia endophenotype. Integrating PPI genes with high-resolution mouse and human brain transcriptomic data, we identified functional and disease coherent co-expression modules through hierarchical clustering and weighted gene co-expression network analysis (WGCNA). In two modules, Schizophrenia risk and mouse PPI genes converged based on telencephalic patterning. The associated neuronal genes were highly expressed in cingulate cortex and hippocampus; implicated in synaptic function and neurotransmission and overlapped with the greatest proportion of rare variants. Concordant neuroanatomical patterning revealed novel core Schizophrenia-relevant genes consistent with the Omnigenic hypothesis of complex traits. Among other genes discussed, the developmental and post-synaptic scaffold TANC2 (Tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2) emerged from both networks as a novel core genetic driver of Schizophrenia altering PPI. Aspects of psychiatric disease comorbidity and phenotypic heterogeneity are also explored. Overall, this study provides a framework and galvanizes the value of mouse preclinical genetics and PPI to prioritize both existing and novel human Schizophrenia candidate genes as druggable targets.

In pursuit of a minimal CHO genome: Establishment of large-scale genome deletions

Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell line for the production of complex therapeutic glycoproteins. As CHO cells have evolved as part of a multicellular organism, they harbor many cellular functions irrelevant for their application as production hosts in industrial bioprocesses. Consequently, CHO cells have been the target for numerous genetic engineering efforts in the past, but a tailored host cell chassis holistically optimized for its specific task in a bioreactor is still missing. While the concept of genome reduction has already been successfully applied to bacterial production cells, attempts to create higher eukaryotic production hosts exhibiting reduced genomes have not been reported yet. Here, we present the establishment and application of a large-scale genome deletion strategy for targeted excision of large genomic regions in CHO cells. We demonstrate the feasibility of genome reduction in CHO cells using optimized CRISPR/Cas9 based experimental protocols targeting large non-essential genomic regions with high efficiency. Achieved genome deletions of non-essential genetic regions did not introduce negative effects on bioprocess relevant parameters, although we conducted the largest reported genomic excision of 864 kilobase pairs in CHO cells so far. The concept presented serves as a directive to accelerate the development of a significantly genome-reduced CHO host cell chassis paving the way for a next generation of CHO cell factories.

The Genomic Shock Hypothesis: Genetic and Epigenetic Alterations of Transposable Elements after Interspecific Hybridization in Plants.

Transposable elements (TEs) are major components of plant genomes with the ability to change their position in the genome or to create new copies of themselves in other positions in the genome. These can cause gene disruption and large-scale genomic alterations, including inversions, deletions, and duplications. Host organisms have evolved a set of mechanisms to suppress TE activity and counter the threat that they pose to genome integrity. These includes the epigenetic silencing of TEs mediated by a process of RNA-directed DNA methylation (RdDM). In most cases, the silencing machinery is very efficient for the vast majority of TEs. However, there are specific circumstances in which TEs can evade such silencing mechanisms, for example, a variety of biotic and abiotic stresses or in vitro culture. Hybridization is also proposed as an inductor of TE proliferation. In fact, the discoverer of the transposons, Barbara McClintock, first hypothesized that interspecific hybridization provides a "genomic shock" that inhibits the TE control mechanisms leading to the mobilization of TEs. However, the studies carried out on this topic have yielded diverse results, showing in some cases a total absence of mobilization or being limited to only some TE families. Here, we review the current knowledge about the impact of interspecific hybridization on TEs in plants and the possible implications of changes in the epigenetic mechanisms.

Genetic components of Escherichia coli involved in its complex prey-predator interaction with Myxococcus xanthus.

Myxococcus xanthus and Escherichia coli represent a well-studied microbial predator-prey pair frequently examined in laboratory settings. While significant progress has been made in comprehending the mechanisms governing M. xanthus predation, various aspects of the response and defensive mechanisms of E. coli as prey remain elusive. In this study, the E. coli MG1655 large-scale chromosome deletion library was screened, and a mutant designated as ME5012 was identified to possess significantly reduced susceptibility to predation by M. xanthus. Within the deleted region of ME5012 encompassing seven genes, the significance of dusB and fis genes in driving the observed phenotype became apparent. Specifically, the deletion of fis resulted in a notable reduction in flagellum production in E. coli, contributing to a certain level of resistance against predation by M. xanthus. Meanwhile, the removal of dusB in E. coli led to diminished inducibility of myxovirescin A production by M. xanthus, accompanied by a slight decrease in susceptibility to myxovirescin A. These findings shed light on the molecular mechanisms underlying the complex interaction between M. xanthus and E. coli in a predatory context.

Long-Term Hematopoietic Dysfunction in Patients with Single Large-Scale Mitochondrial DNA Deletion Syndromes

Pearson syndrome (PS) is the only single large-scale mitochondrial DNA deletion syndrome (SLSMDs) presenting substantial hematologic manifestations, usually a severe though transient cytopenia of childhood. Recently, progression to myeloid malignancies has been described among PS patients. Older PS patients, as well as patients with SLSMDs presenting with Kearns-Sayre syndrome (KSS) or with progressive external ophthalmoplegia (PEO), do not display hematologic abnormalities. Since the underlying mitochondrial DNA deletion is similar in all SLSMDs, we hypothesized that bone marrow (BM) dysfunction is a common and persistent manifestation in these patients. We studied 16 patients with molecular-confirmed SLSMDs: 13 with PS, and three with KSS. The median age at diagnosis was 25 months (range 2-136 months). Seventy-five percent of patients presented with cytopenia in infancy, and 56% required blood products. Resolution of cytopenia occurred in 92% of this subgroup, at a median age of 24 months (range 12-39 months). BM studies were performed in all patients, and 56% of patients had multiple BM evaluations. All BM biopsies examined showed signs of marrow dysfunction, including in patients without a history of cytopenia. Median BM cellularity was 50% (range 30-80%) at a median age of 5 years. BM samples showed a paucity of precursor myeloid cells, signs of dyserythropoiesis, and abnormal or missing megakaryocytes. We report low hematopoietic stem and progenitor cell functionality in patients with SLSMDs: The median CD34+ count was 0.68% (range 0.27-1.92%, expected range 1-2%). BM colony forming unit (CFU) capacity was reduced in SLSMD patients: the median CFU in patients was 280 per 10 6 cells compared to 1090 per 10 6 cells in healthy controls (p=0.004). Conventional cytogenetic analysis was done on 10 patients (62%), and FISH analysis was done on 15 patients (93%). Monosomy 7 was identified in seven patients (44%): in one patient the monosomy 7 resolved in a follow-up BM assay. Another patient with signs of myelodysplasia and 30% of the BM cells having monosomy 7 was referred to an allogeneic bone marrow transplantation. To conclude, long-term BM dysfunction is evident in all patients with SLSMD and includes a risk of developing clonal evolution and monosomy 7. This data, combined with other recent publications, highlights the ongoing marrow failure and suggest that SLSMD syndromes should be included under inherited BM failure syndromes.

The clinical, myopathological, and genetic analysis of 155 Chinese mitochondrial ophthalmoplegia patients with mitochondrial DNA single large deletions.

Progressive external ophthalmoplegia (PEO) is a common subtype of mitochondrial encephalomyopathy. The study aimed to investigate the relationship between mitochondrial DNA (mtDNA) abnormalities, muscle pathology, and clinical manifestations in Chinese patients with single large-scale mtDNA deletion presenting with PEO. This is a retrospective single-center study. Patients with PEO who had a single large deletion in mitochondrial DNA were included in this study. The associations were analyzed between mtDNA deletion patterns, myopathological changes, and clinical characteristics. In total, 155 patients with mitochondrial PEO carrying single large-scale mtDNA mutations were enrolled, including 137 chronic progressive external ophthalmoplegia (CPEO) and 18 Kearns-Sayre syndrome (KSS) patients. The onset ages were 9.61 ± 4.12 in KSS and 20.15 ± 9.06 in CPEO. The mtDNA deletions ranged from 2225 bp to 9131 bp, with m.8470_13446del being the most common. The KSS group showed longer deletions than the CPEO group (p = 0.004). Additionally, a higher number of deleted genes encoding respiratory chain complex subunits (p = 0.001) and tRNA genes (p = 0.009) were also observed in the KSS group. A weak negative correlation between the mtDNA deletion size and ages of onset (p < 0.001, r = -0.369) was observed. The proportion of ragged red fibers, ragged blue fibers, and cytochrome c negative fibers did not correlate significantly with onset ages (p > 0.05). However, a higher percentage of abnormal muscle fibers corresponds to an increased prevalence of exercise intolerance, limb muscle weakness, dysphagia, and cerebellar ataxia. We reported a large Chinese cohort consisting of mitochondrial PEO patients with single large-scale mtDNA deletions. Our results demonstrated that the length and locations of mtDNA deletions may influence onset ages and clinical phenotypes. The severity of muscle pathology could not only indicate diagnosis but also may be associated with clinical manifestations beyond the extraocular muscles.

Genomics of predictive radiation mutagenesis in oilseed rape: modifying seed oil composition.

Rapeseed is a crop of global importance but there is a need to broaden the genetic diversity available to address breeding objectives. Radiation mutagenesis, supported by genomics, has the potential to supersede genome editing for both gene knockout and copy number increase, but detailed knowledge of the molecular outcomes of radiation treatment is lacking. To address this, we produced a genome re-sequenced panel of 1133 M2 generation rapeseed plants and analysed large-scale deletions, single nucleotide variants and small insertion-deletion variants affecting gene open reading frames. We show that high radiation doses (2000 Gy) are tolerated, gamma radiation and fast neutron radiation have similar impacts and that segments deleted from the genomes of some plants are inherited as additional copies by their siblings, enabling gene dosage decrease. Of relevance for species with larger genomes, we showed that these large-scale impacts can also be detected using transcriptome re-sequencing. To test the utility of the approach for predictive alteration of oil fatty acid composition, we produced lines with both decreased and increased copy numbers of Bna.FAE1 and confirmed the anticipated impacts on erucic acid content. We detected and tested a 21-base deletion expected to abolish function of Bna.FAD2.A5, for which we confirmed the predicted reduction in seed oil polyunsaturated fatty acid content. Our improved understanding of the molecular effects of radiation mutagenesis will underpin genomics-led approaches to more efficient introduction of novel genetic variation into the breeding of this crop and provides an exemplar for the predictive improvement of other crops.

From correlation to causation using directed topological overlap matrix: Applications in genomics.

Most causal discovery tools assume the local causal Markov condition. However, the theoretical assumptions that underlie the local causal Markov condition are often not met in practice. This is especially marked in genomics, where the unwanted presence of measurement errors, averaging effects, and feedback loops significantly undermine the legitimacy of the local causal Markov condition. Furthermore, these causal discovery algorithms require very large samples, orders above what is often available. In this paper, relaxing the local causal Markov condition and using Reichenbach's common cause principle instead, we present a more flexible approach to causal discovery, the directed topological overlap matrix (DTOM). DTOM is robust w.r.t the presence of measurement errors, averaging effects, feedback loops, and is significantly more sample efficient. We study the utility of DTOM for discovering causal relations in biological data using three real gene expression data-sets. We first examine if DTOM can help distinguish the Myostatin mutation in the Piedmontese cattle by contrasting the muscle transcriptomes of the Piedmontese and Wagyu crosses: the Myostatin mutation is the cause of the double-muscling the Piedmontese cattle are famous for. We then consider a large-scale gene deletion study in yeast. We show that DTOM allows us to distinguish the deleted gene in a sample knowing only the set of differentially expressed genes in that sample. We then examine the progression of Alzheimer's disease (AD) under the lens of DTOM. The genes implicated as having a causal role in the progression of AD by our DTOM analysis were significantly enriched in cellular components that had been repeatedly implicated in the progression of AD.

Novel Homozygous Variants of SLC13A5 Expand the Functional Heterogeneity of a Homogeneous Syndrome of Early Infantile Epileptic Encephalopathy

BackgroundEarly infantile epileptic encephalopathy 25 (EIEE25) is a distinct type of neonatal epileptic encephalopathy caused by autosomal recessive mutations in the SLC13A5 gene. SLC13A5 encodes a transmembrane sodium/citrate cotransporter required for regulating citrate entry into cells. MethodsFour families with recessively inherited epileptic encephalopathy were sequenced by clinically accredited laboratories using commercially available epilepsy gene panels. Patients were examined by a neurologist and were clinically diagnosed with infantile epileptic encephalopathy. ResultsWe present four families with global developmental delay, intellectual disability, and defective tooth development with four novel homozygous mutations in SLC13A5. The neurological examination showed spastic quadriplegia with increased deep tendon reflexes. Brain magnetic resonance imaging showed nonspecific signal abnormality of the bilateral hemispheric white matter. Despite similar clinical features, the conditions were based on different molecular mechanisms acting on SLC13A5 (abnormal splicing, large-scale deletions, and tandem-residue insertion). ConclusionsOur results extend the landscape of autosomal recessive inherited homozygous mutations in SLC13A5 that cause a distinctive syndrome of severe neonatal epileptic encephalopathy. Our observations confirm the homogeneity of epileptic encephalopathy and dental abnormalities as a distinct clinical marker for EIEE25 despite the heterogeneous functional and mutational background.