Entire Chromosome Research Articles

Frequent horizontal chromosome transfer between asexual fungal insect pathogens

Entire chromosomes are typically only transmitted vertically from one generation to the next. The horizontal transfer of such chromosomes has long been considered improbable, yet gained recent support in several pathogenic fungi where it may affect the fitness or host specificity. To date, it is unknown how these transfers occur, how common they are, and whether they can occur between different species. In this study, we show multiple independent instances of horizontal transfers of the same accessory chromosome between two distinct strains of the asexual entomopathogenic fungus Metarhizium robertsii during experimental co-infection of its insect host, the Argentine ant. Notably, only the one chromosome-but no other-was transferred from the donor to the recipient strain. The recipient strain, now harboring the accessory chromosome, exhibited a competitive advantage under certain host conditions. By phylogenetic analysis, we further demonstrate that the same accessory chromosome was horizontally transferred in a natural environment between M. robertsii and another congeneric insect pathogen, Metarhizium guizhouense. Hence, horizontal chromosome transfer is not limited to the observed frequent events within species during experimental infections but also occurs naturally across species. The accessory chromosome that was transferred contains genes that may be involved in its preferential horizontal transfer or support its establishment. These genes encode putative histones and histone-modifying enzymes, as well as putative virulence factors. Our study reveals that both intra- and interspecies horizontal transfer of entire chromosomes is more frequent than previously assumed, likely representing a not uncommon mechanism for gene exchange.

Segmental duplications drive the evolution of accessory regions in a major crop pathogen.

Many pathogens evolved compartmentalized genomes with conserved core and variable accessory regions (ARs) that carry effector genes mediating virulence. The fungal plant pathogen Fusarium oxysporum has such ARs, often spanning entire chromosomes. The presence of specific ARs influences the host range, and horizontal transfer of ARs can modify the pathogenicity of the receiving strain. However, how these ARs evolve in strains that infect the same host remains largely unknown. We defined the pan-genome of 69 diverse F. oxysporum strains that cause Fusarium wilt of banana, a significant constraint to global banana production, and analyzed the diversity and evolution of the ARs. Accessory regions in F. oxysporum strains infecting the same banana cultivar are highly diverse, and we could not identify any shared genomic regions and in planta-induced effectors. We demonstrate that segmental duplications drive the evolution of ARs. Furthermore, we show that recent segmental duplications specifically in accessory chromosomes cause the expansion of ARs in F. oxysporum. Taken together, we conclude that extensive recent duplications drive the evolution of ARs in F. oxysporum, which contribute to the evolution of virulence.

New insights into the systematics of the afrotropical Amblyomma marmoreum complex (Acari: Ixodidae) and the genome of a novel Rickettsia africae strain using morphological and metagenomic approaches

The Amblyomma marmoreum complex includes afrotropical species, such as Amblyomma sparsum, a three-host tick that parasitizes reptiles, birds, and mammals, and is a recognized vector of Ehrlichia ruminantium. However, the lack of morphological, genetic and ecological data on A. sparsum has caused considerable confusion in its identification. In this study, we used microscopy and metagenomic approaches to analyze A. sparsum ticks collected from a puff adder snake (Bitis arietans) in southwest Senegal (an endemic rickettsioses area) in order to supplement previous morphological descriptions, provide novel genomic data for the A. marmoreum complex, and describe the genome of a novel spotted fever group Rickettsia strain. Based on stereoscope and scanning electron microscopy (SEM) morphological evaluations, we provide high-quality images and new insights about punctation and enameling in the adult male of A. sparsum to facilitate identification for future studies. The metagenomic approach allowed us assembly the complete mitochondrial genome of A. sparsum, as well as the nearly entire chromosome and complete plasmid sequences of a novel Rickettsia africae strain. Phylogenomic analyses demonstrated a close relationship between A. sparsum and Amblyomma nuttalli for the first time and confirmed the position of A. sparsum within the A. marmoreum complex. Our results provide new insights into the systematics of A. sparsum and A. marmoreum complex, as well as the genetic diversity of R. africae in the Afrotropical region. Future studies should consider the possibility that A. sparsum may be a vector for R. africae.

Clinical, genetic profile and therapy evaluation of 11 Chinese pediatric patients with Fanconi-Bickel syndrome

BackgroundFanconi-Bickel syndrome (FBS) is a rare autosomal recessive disorder characterized by impaired glucose and galactose utilization as well as proximal renal tubular dysfunction.MethodsClinical, biochemical, genetic, treatment, and follow-up data for 11 pediatric patients with FBS were retrospectively analysed.ResultsHepatomegaly (10/11), short stature (10/11) and hypophosphataemic rickets (7/11) were the most common initial symptoms. At diagnosis, all patients had decreased fasting blood glucose (FBG), plasma bicarbonate (HCO3−) and serum phosphorus, as well as elevated liver transaminases, alkaline phosphatase (AKP) and proximal renal tubular dysfunction. Two infant patients were misdiagnosed with transient neonatal diabetes mellitus. After therapy with uncooked cornstarch and conventional rickets treatment, remission of hepatomegaly was observed in all patients, with significant improvements in pre-prandial blood glucose, liver transaminases, triglyceride, plasma HCO3− and AKP (p < 0.05). At the last follow-up, 5/7 patients with elevated AKP had nephrocalcinosis. The mean height standard deviation score (Ht SDS) of eight patients with regular treatment increased from − 4.1 to -3.5 (p = 0.02). Recombinant human growth hormone (rhGH) was administered to 4/9 patients, but their Ht SDS did not improve significantly (p = 0.13). Fourteen variants of the SLC2A2 gene were identified, with six being novel, among which one was recurrent: c.1217T > G (p.L406R) (allele frequency: 4/22, 18%). Patients with biallelic missense variants showed milder metabolic acidosis than those with null variants. Two of five patients from nonconsanguineous families with rare homozygous variations showed 5.3 Mb and 36.6 Mb of homozygosity surrounding the variants, respectively; a region of homozygosity (ROH) involving the entire chromosome 3 covering the SLC2A2 gene, suggesting uniparental disomy 3, was detected in one patient.ConclusionsEarly diagnosis of FBS is difficult due to the heterogeneity of initial symptoms. Although short stature is a major issue of treatment for FBS, rhGH is not recommended in FBS patients who have normal GH stimulation tests. Patients with biallelic null variants may require alkali supplementation since urine bicarbonate loss is genetically related. ROH is a mechanism for rare homozygous variants of FBS in nonconsanguineous families.

Pediatric spinal ependymoma with chromothripsis of chromosome 6: a case report and review of the literature

BackgroundEpendymomas are the third most common central nervous system tumor in the pediatric population; however, spinal ependymomas in children are rare. Ependymomas affecting the spinal cord most frequently occur in adults of 20–40 years of age. The current World Health Organization classification system for ependymomas is now composed of ten different entities based on histopathology, location, and molecular studies, with evidence that the new classification system more accurately predicts clinical outcomes.Case presentationWe present the case of a 16-year-old Caucasian female patient with a history of type 2 neurofibromatosis with multiple schwannomas, meningioma, and spinal ependymoma. Chromosome analysis of the harvested spinal ependymoma tumor sample revealed a 46,XX,−6,+7,−22,+mar[16]/46,XX[4] karyotype. Subsequent OncoScan microarray analysis of the formalin-fixed paraffin-embedded tumor sample confirmed + 7, −22 and clarified that the marker chromosome represents chromothripsis of the entire chromosome 6 with more than 100 breakpoints. Fluorescent in situ hybridization and microarray analysis showed no evidence of MYCN amplification. The final integrated pathology diagnosis was spinal ependymoma (central nervous system World Health Organization grade 2 with no MYCN amplification.ConclusionThis case adds to the existing literature of pediatric patients with spinal ependymomas and expands the cytogenetic findings that may be seen in patients with this tumor type. This case also highlights the value of cytogenetics and microarray analysis in solid tumors to provide a more accurate molecular diagnosis.

Aneuploidy and complex genomic rearrangements in cancer evolution.

Mutational processes that alter large genomic regions occur frequently in developing tumors. They range from simple copy number gains and losses to the shattering and reassembly of entire chromosomes. These catastrophic events, such as chromothripsis, chromoplexy and the formation of extrachromosomal DNA, affect the expression of many genes and therefore have a substantial effect on the fitness of the cells in which they arise. In this review, we cover large genomic alterations, the mechanisms that cause them and their effect on tumor development and evolution.

Conventional cytogenetics and microsatellite chromosomal distribution in social wasp Mischocyttarus cassununga (Ihering, 1903) (Vespidae, Polistinae, Mischocyttarini).

Cytogenetics has allowed the investigation of chromosomal diversity and repetitive genomic content in wasps. In this study, we characterized the karyotype of the social wasp Mischocyttarus cassununga using conventional cytogenetics and chromosomal mapping of repetitive sequences. This study was undertaken to extend our understanding of the genomic organization of repetitive DNA in social wasps and is the first molecular cytogenetic insight into the genus Mischocyttarus. The karyotype of M.cassununga had a chromosome number of 2n=64 for females and n=32 for males. Constitutive heterochromatin exhibited three distribution patterns: centromeric and pericentromeric regions along the smaller arms and extending almost the entire chromosome. The major ribosomal DNA sites were located on chromosome pair in females and one chromosome in males. Positive signals for the microsatellite probes (GA)n and (GAG)n were observed in the euchromatic regions of all chromosomes. The microsatellites, (CGG)n, (TAT)n, (TTAGG)n, and (TCAGG)n were not observed in any region of the chromosomes. Our results contrast with those previously obtained for Polybia fastidiosuscula, which showed that the microsatellites (GAG)n, (CGG)n, (TAT)n, (TTAGG)n, and (TCAGG)n are located predominantly in constitutive heterochromatin. This suggests variations in the diversity and chromosomal organization of repetitive sequences in the genomes of social wasps.

Borrelia nietonii sp. nov.: Relapsing Fever Spirochetes Transmitted By the Tick Ornithodoros hermsi Designated Previously as Borrelia hermsii Genomic Group II.

Background: The taxonomic status of the relapsing fever spirochete Borrelia hermsii in western North America was established in 1942 and based solely on its specific association with the soft tick vector Ornithodoros hermsi. Multilocus sequence typing (MLST) of the 16S rRNA, flaB, gyrB, glpQ, and 16S-23S rRNA intergenic spacer of B. hermsii isolates collected over many years from various geographic locations and biological sources identified two distinct clades designated previously as B. hermsii Genomic Group I (GGI) and Genomic Group II (GGII). To better assess the taxonomic relationship of these two genomic groups to each other and other species of Borrelia, DNA sequences of the entire linear chromosome were determined. Materials and Methods: Genomic DNA samples were prepared from 11 spirochete isolates grown in Barbour-Stoenner-Kelly-H medium. From these preparations, DNA sequences of the entire linear chromosome of two isolates of B. hermsii belonging to each genomic group and seven additional species were determined. Results: Chromosomal sequences of four isolates of B. hermsii contained 919,212 to 922,307 base pairs. DNA sequence identities between the two genomic groups of B. hermsii were 95.86-95.99%, which were more divergent than chromosomal sequences comparing Borrelia parkeri and Borrelia turicatae (97.13%), Borrelia recurrentis and Borrelia duttonii (97.07%), and Borrelia crocidurae and B. duttonii (97.09%). The 3' end of the chromosome of the two GGII isolates also contained a unique intact oppA gene absent from all other species examined. Conclusion: Previous MLST and the chromosomal sequences presented herein support the division of the B. hermsii species complex into two species, B. hermsii sensu stricto ( = GGI) and Borrelia nietonii sp. nov. ( = GGII). We name this unique relapsing fever spirochete in honor of our late friend and colleague Dr. Nathan Nieto for his outstanding contributions to our understanding of tick-borne relapsing fever.

Structural variants in Lewy body dementia and frontotemporal dementia spectrum

AbstractBackgroundStructural variants range from simple loss or gain of genetic material to complex events that restructure entire chromosomes. This heterogeneity coupled with variant size greater than sequencing read‐length makes structural variant mapping from short‐read sequencing data difficult and error‐prone. Consequently, the role of structural variants is poorly explored in many phenotypes, including dementia.MethodWe used the GATK‐SV pipeline that combines four external structural variant mapping algorithms with machine‐learning to build high‐quality consensus structural variant calls. We applied GATK‐SV to short‐read whole‐genome sequencing data of 2,601 frontotemporal dementia‐amyotrophic lateral sclerosis spectrum (FTD/ALS) patients, 2,612 Lewy body dementia (LBD) patients, and 4,132 neurologically unaffected participants.ResultAfter stringent quality‐control, we performed genome‐wide association studies on 2,307 ALS/FTD patients versus 3,677 controls with 4,699 common structural variants and 2,355 LBD patients versus 3,700 controls with 4,889 common structural variants. In the FTD/ALS cohort, we identified well‐known risk variants at the C9orf72 (p‐value = 4.99×10‐18, OR = 14.47, 95% CI = 7.90–26.49) and MAPT loci (p‐value = 3.48×10‐6, OR = 0.77, 95% CI = 0.68–0.86) corroborating the ability of our pipeline to identify disease‐associated structural variants. Moreover, we discovered, replicated, and validated TPCN1 as a novel risk locus for LBD (p‐value = 9.18×10‐6, OR = 1.43, 95% CI = 1.22–1.67). Further, we identified rare exonic variants with known pathogenic or likely pathogenic effects, such as an SNCA gene duplication in LBD and a deletion of exons 1 and 2 in CHCHD10 in FTD/ALS. We additionally observed structural variants in regulatory regions, e.g. a deletion of a key PSEN2 enhancer, which resulted in an approximate 0.70 fold‐change in PSEN2 expression in LBD. Finally, we built an interactive web app where structural variants can be visualized and explored (https://ndru-ndrs-lng-nih.shinyapps.io/non_ad_dementias_sv_app/).ConclusionCommon and rare structural variants play a role in LBD and FTD/ALS. Our structural variant resource is publicly available and can be used to further explore the role of structural variants in LBD and FTD/ALS.

Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms

Myeloproliferative Neoplasms (MPNs) are a diverse group of clonal hematopoietic disorders originating from a single hematopoietic stem cell, causing an excessive production of mature blood cells. Classic forms of MPNs include polycythemia vera, essential thrombocythemia, and myelofibrosis. The most common mutation is a gain-of-function point mutation in the JAK2 gene, known as JAK2V617F. This mutation leads to constant activation of the JAK-STAT signaling pathway, resulting in the overgrowth of MPN cells. Furthermore, the variant allele frequency (VAF) of JAK2V617F has a significant impact on the severity and phenotype of the disease. Alongside many described genetic mutations, cytogenetic abnormalities are commonly observed in MPNs, particularly involving chromosome 9. As the JAK2 gene is located on the short arm of this chromosome, we hypothesized that chromosome 9 copy number abnormalities might be a disease modifier in JAK2V617F-mutant MPN patients. To characterize the biological effects of chromosome 9 copy number abnormalities on JAK2-mutated MPN cells, we analyzed circulating CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as monocytes and granulocytes from 32 MPN patients. Through Next Generation Sequencing, we categorized the patients into three main groups based on JAK2 mutation and copy number status: patients with two copies of the JAK2 gene with either heterozygous (VAF between 20% and 60%; n=10) or homozygous (VAF &amp;gt; 60% n=10) JAK2V617F mutation, or patients carrying mutant JAK2 and gene amplification (n=12). In-depth analysis of JAK2-amplified patients revealed that the amplification involved the entire chromosome 9p, thus including other gene loci like CD274, which encodes programmed death-ligand 1 (PD-L1). Further investigation of the order-of-events and clonal hierarchies through droplet digital PCR on CD34+ cell-derived colonies showed that most colonies of 9p-duplicated patients had three copies of JAK2, with 2 out of 3 alleles harboring the JAK2 mutation and that point mutations are frequently the initial pathogenic event in clonal evolution, followed by amplification of the JAK2-mutated allele. Functionally, CD34+ cells from +9p patients displayed high clonogenicity and gave rise to a greater number of primitive colonies (Colony Forming Unit-Granulocyte, Erythrocyte, Monocyte, Megakaryocyte, CFU-GEMM). As JAK2 hyperactivation had been previously reported to lead to increased PD-L1 expression, we further explored the functional significance of CD274 amplification in +9p patients. Our analysis showed increased PD-L1 messenger RNA and protein levels in +9p patient CD14+ monocytes compared to those from patients carrying only two copies of chromosome 9. Moreover, immunofluorescence analysis demonstrated significant re-localization of PD-L1 to the cytoplasmic membrane in monocytes from +9p patients, but not in JAK2V617F-homozygous patients (panel A). Increased levels of PD-L1, an immune checkpoint known to curb T cell activation, led us to analyze the T cell compartment, which resulted enriched in CD3+/CD8+/CD57-/PD-1+ exhausted T-cells in +9p patients compared to other MPN patients and healthy donors (panel B). In conclusion, our comprehensive characterization of the molecular interplay between JAK2V617F and chromosome 9 alterations, along with their immunological implications due to PD-L1 hyperactivation, fills a critical knowledge gap and provides valuable insights into the disease progression of 9p-MPNs. Further analysis is ongoing to explore the associations between 9p duplication and hematological parameters in 9p-MPN patients for a better understanding of the clinical implications of this genetic abnormality in MPNs.

PATH-15. GLIOBLASTOMA, IDH-WILDTYPE WITHOUT KEY MOLECULAR FEATURES.

Abstract Molecular alterations that predict aggressive behavior of astrocytic tumors include EGFR amplification, TERT promoter (pTERT) mutations, gain of chromosome 7 and loss of chromosome 10. During January 2020 and February 2023, we encountered 21 cases of pathologically diagnosed glioblastoma, IDH-wildtype without the above-mentioned molecular characteristics. In this study, we tried to characterize these tumors. For all cases, genomic DNA was extracted from fresh frozen tissue. Using extracted DNA, to analyze the status of IDH mutation, 1p/19q codeletion, pTERT mutation, EGFR gene amplification, combined gain of entire chromosome 7, and loss of entire chromosome 10 (+7/− 10), whole exome sequencing, Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) was performed. In addition, gene panel testing was performed for suspected familial cases. Among 21 cases, 2 harbored histone G34 mutation and was diagnosed as diffuse hemispheric gliomas, H3 G34-mutant. Among the rest 19 patients, CDKN2A/B homozygous deletions were detected in 7. Three of the rest 12 patients were diagnosed as patients with Li Fraumeni syndrome. Glioblastoma IDH-wildtype CNS WHO grade 4 typically presents necrosis and/or microvascular proliferation. Molecular alterations including EGFR amplification, pTERT mutations, gain of chromosome 7 and loss of chromosome 10 are key features that characterize GBM IDH-wildtype. However, some of the pathologically diagnosed GBM, do not carry these gene alterations. Among those, there existed cases harboring CDKN2A/B homozygous deletions or H3 G34 mutations, and cases suffering Li Fraumeni syndrome.

Trisomies Reorganize Human 3D Genome.

Trisomy is the presence of one extra copy of an entire chromosome or its part in a cell nucleus. In humans, autosomal trisomies are associated with severe developmental abnormalities leading to embryonic lethality, miscarriage or pronounced deviations of various organs and systems at birth. Trisomies are characterized by alterations in gene expression level, not exclusively on the trisomic chromosome, but throughout the genome. Here, we applied the high-throughput chromosome conformation capture technique (Hi-C) to study chromatin 3D structure in human chorion cells carrying either additional chromosome 13 (Patau syndrome) or chromosome 16 and in cultured fibroblasts with extra chromosome 18 (Edwards syndrome). The presence of extra chromosomes results in systematic changes of contact frequencies between small and large chromosomes. Analyzing the behavior of individual chromosomes, we found that a limited number of chromosomes change their contact patterns stochastically in trisomic cells and that it could be associated with lamina-associated domains (LAD) and gene content. For trisomy 13 and 18, but not for trisomy 16, the proportion of compacted loci on a chromosome is correlated with LAD content. We also found that regions of the genome that become more compact in trisomic cells are enriched in housekeeping genes, indicating a possible decrease in chromatin accessibility and transcription level of these genes. These results provide a framework for understanding the mechanisms of pan-genome transcription dysregulation in trisomies in the context of chromatin spatial organization.

An automatic progressive chromosome segmentation approach using deep learning with traditional image processing.

The fully automatic chromosome analysis system plays an important role in the detection of genetic diseases, which in turn can reduce the diagnosis burden for cytogenetic experts. Chromosome segmentation is a critical step for such a system. However, due to the non-rigid structure of chromosomes, chromosomes may curve in any direction, and two or more chromosomes may touch or overlap to form unpredictable chromosome clusters in metaphase chromosome images, leading to automatic chromosome segmentation as a challenge. In this paper, we propose an automatic progressive segmentation approach to perform the entire metaphase chromosome image segmentation using deep learning with traditional image processing. It follows three stages. In the first stage, thresholding-based and geometric-based methods are employed to divide all chromosomes as single ones and chromosome clusters. To tackle the segmentation for unpredictable chromosome clusters, we first present a new chromosome cluster identification network named CCI-Net to classify all chromosome clusters into different types in the second stage, and then in the third stage, we combine traditional image processing with deep CNNs to accomplish chromosome instance segmentation from different types of clusters. Evaluation results on a clinical dataset of 1148 metaphase chromosome images show that the proposed automatic progressive segmentation method achieves 94.60% chromosome cluster identification accuracy and 99.15% instance segmentation accuracy. The experimental results exhibit that our proposed approach can effectively identify chromosome clusters and successfully perform fully automatic chromosome segmentation.

Differential Spreading of Microsatellites in Holocentric Chromosomes of Chagas Disease Vectors: Genomic and Evolutionary Implications.

This study focused on analyzing the distribution of microsatellites in holocentric chromosomes of the Triatominae subfamily, insect vectors of Chagas disease. We employed a non-denaturing FISH technique to determine the chromosomal distribution of sixteen microsatellites across twenty-five triatomine species, involving five genera from the two principal tribes: Triatomini and Rhodniini. Three main hybridization patterns were identified: strong signals in specific chromosomal regions, dispersed signals dependent on microsatellite abundance and the absence of signals in certain chromosomal regions or entire chromosomes. Significant variations in hybridization patterns were observed between Rhodniini and Triatomini species. Rhodniini species displayed weak and scattered hybridization signals, indicating a low abundance of microsatellites in their genomes. In contrast, Triatomini species exhibited diverse and abundant hybridization patterns, suggesting that microsatellites are a significant repetitive component in their genomes. One particularly interesting finding was the high abundance of GATA repeats, and to a lesser extent AG repeats, in the Y chromosome of all analyzed Triatomini species. In contrast, the Y chromosome of Rhodniini species did not show enrichment in GATA and AG repeats. This suggests that the richness of GATA repeats on the Y chromosome likely represents an ancestral trait specific to the Triatomini tribe. Furthermore, this information can be used to elucidate the evolutionary relationships between Triatomini and other groups of reduviids, contributing to the understanding of the subfamily's origin. Overall, this study provides a comprehensive understanding of the composition and distribution of microsatellites within Triatominae genomes, shedding light on their significance in the evolutionary processes of these species.

Adaptive sampling for nanopore direct RNA-sequencing.

Nanopore long-read sequencing enables real-time monitoring and controlling of individual nanopores. This allows us to enrich or deplete specific sequences in DNA sequencing in a process called "adaptive sampling." So far, adaptive sampling (AS) was not applicable to the direct sequencing of RNA. Here, we show that AS is feasible and useful for direct RNA sequencing (DRS), which has its specific technical and biological challenges. Using a well-controlled in vitro transcript-based model system, we identify essential characteristics and parameter settings for AS in DRS, as the superior performance of depletion over enrichment. Here, the efficiency of depletion is close to the theoretical maximum. Additionally, we demonstrate that AS efficiently depletes specific transcripts in transcriptome-wide sequencing applications. Specifically, we applied our AS approach to poly(A)-enriched RNA samples from human-induced pluripotent stem cell-derived cardiomyocytes and mouse whole heart tissue and show efficient 2.5- to 2.8-fold depletion of highly abundant mitochondrial-encoded transcripts. Finally, we characterize depletion and enrichment performance for complex transcriptome subsets, that is, at the level of the entire Chromosome 11, proving the general applicability of direct RNA AS. Our analyses provide evidence that AS is especially useful to enable the detection of lowly expressed transcripts and reduce the sequencing of highly abundant disturbing transcripts.

Oligonucleotide Fluorescence In Situ Hybridization: An Efficient Chromosome Painting Method in Plants.

Fluorescence in situ hybridization (FISH) is an indispensable technique for studying chromosomes in plants. However, traditional FISH methods, such as BAC, rDNA, tandem repeats, and distributed repetitive sequence probe-based FISH, have certain limitations, including difficulties in probe synthesis, low sensitivity, cross-hybridization, and limited resolution. In contrast, oligo-based FISH represents a more efficient method for chromosomal studies in plants. Oligo probes are computationally designed and synthesized for any plant species with a sequenced genome and are suitable for single and repetitive DNA sequences, entire chromosomes, or chromosomal segments. Furthermore, oligo probes used in the FISH experiment provide high specificity, resolution, and multiplexing. Moreover, oligo probes made from one species are applicable for studying other genetically and taxonomically related species whose genome has not been sequenced yet, facilitating molecular cytogenetic studies of non-model plants. However, there are some limitations of oligo probes that should be considered, such as requiring prior knowledge of the probe design process and FISH signal issues with shorter probes of background noises during oligo-FISH experiments. This review comprehensively discusses de novo oligo probe synthesis with more focus on single-copy DNA sequences, preparation, improvement, and factors that affect oligo-FISH efficiency. Furthermore, this review highlights recent applications of oligo-FISH in a wide range of plant chromosomal studies.

Detection of Primary DNA Lesions by Transient Changes in Mating Behavior in Yeast Saccharomyces cerevisiae Using the Alpha-Test.

Spontaneous or induced DNA lesions can result in stable gene mutations and chromosomal aberrations due to their inaccurate repair, ultimately resulting in phenotype changes. Some DNA lesions per se may interfere with transcription, leading to temporary phenocopies of mutations. The direct impact of primary DNA lesions on phenotype before their removal by repair is not well understood. To address this question, we used the alpha-test, which allows for detecting various genetic events leading to temporary or hereditary changes in mating type α→a in heterothallic strains of yeast Saccharomyces cerevisiae. Here, we compared yeast strains carrying mutations in DNA repair genes, mismatch repair (pms1), base excision repair (ogg1), and homologous recombination repair (rad52), as well as mutagens causing specific DNA lesions (UV light and camptothecin). We found that double-strand breaks and UV-induced lesions have a stronger effect on the phenotype than mismatches and 8-oxoguanine. Moreover, the loss of the entire chromosome III leads to an immediate mating type switch α→a and does not prevent hybridization. We also evaluated the ability of primary DNA lesions to persist through the cell cycle by assessing the frequency of UV-induced inherited and non-inherited genetic changes in asynchronous cultures of a wild-type (wt) strain and in a cdc28-4 mutant arrested in the G1 phase. Our findings suggest that the phenotypic manifestation of primary DNA lesions depends on their type and the stage of the cell cycle in which it occurred.

The Fluorescence in situ hybridization (FISH): types and application: a review

The macromolecule identification method known as fluorescence in situ hybridization (FISH) is regarded as a recent development in the study of cytology. It was first created as a physical mapping method to identify genes inside chromosomes. Later, biological and medical studies benefited from the precision and adaptability of FISH. This attractive method offers a level of resolution in between chromosomal studies and DNA analysis. A hybridizing DNA probe used in FISH can either be directly or indirectly labeled. Fluorescent nucleotides are employed in direct labeling, whereas reporter molecules used in indirect labeling are afterwards recognized through affinity molecules, fluorescent antibodies, or other means. An excessively high number of chromosomes in a cell, unique gene fusions, or the loss of a chromosomal segment or an entire chromosome are examples of genetic disorders that can be detected with FISH. It is also used in a variety of scientific projects, including gene mapping and the discovery of fresh oncogenes. This article examines the FISH idea, its use, and its benefits in medical research.

Disruption of the standard kinetochore in holocentric Cuscuta species

The segregation of chromosomes depends on the centromere. Most species are monocentric, with the centromere restricted to a single region per chromosome. In some organisms, the monocentric organization changed to holocentric, in which the centromere activity is distributed over the entire chromosome length. However, the causes and consequences of this transition are poorly understood. Here, we show that the transition in the genus Cuscuta was associated with dramatic changes in the kinetochore, a protein complex that mediates the attachment of chromosomes to microtubules. We found that in holocentric Cuscuta species, the KNL2 genes were lost; the CENP-C, KNL1, and ZWINT1 genes were truncated; the centromeric localization of CENH3, CENP-C, KNL1, MIS12, and NDC80 proteins was disrupted; and the spindle assembly checkpoint (SAC) degenerated. Our results demonstrate that holocentric Cuscuta species lost the ability to form a standard kinetochore and do not employ SAC to control the attachment of microtubules to chromosomes.

Abstract LB080: SAVANA: a computational method to characterize structural variation in human cancer genomes using nanopore sequencing

Abstract Whole-genome sequencing (WGS) of human cancers has revealed that structural variation, which refers to the rearrangement of the genome leading to the deletion, amplification of reshuffling of DNA segments ranging from a few hundred bp to entire chromosomes, is a key mutational process in cancer evolution. Notably, pan-cancer analyses have revealed that both simple and complex forms of structural variation are pervasive across diverse human cancers, and often underpin drug resistance and metastasis. To date, the study of cancer genomes has relied on the analysis of short-read WGS on the dominant Illumina platform, which generates short, highly-accurate reads of 100-300bp that allow the study of point mutations at high resolution. However, detection of structural variants (SVs) using short reads is limited, as breakpoints falling in repetitive regions cannot be reliably mapped to the human genome. As a result, our understanding of the patterns and mechanisms underpinning structural variation in cancer genomes remains incomplete. In contrast to short-read sequencing, long-read sequencing technologies, such as Oxford Nanopore and PacBio, permit continuous reading of individual DNA molecules over 10 kilobases, thus providing unparalleled information to resolve SVs in repetitive regions and complex genome rearrangements. However, novel bioinformatics methods that account for the higher error rate of long-read methods are needed to take advantage of their capabilities for cancer genome analysis. Here, we present SAVANA, a novel structural variant caller for long-read sequencing data specifically designed for the analysis of cancer genomes. To identify both somatic and germline SVs, SAVANA takes as input long-read WGS data from a tumor and normal sample pair. SAVANA scans sequencing reads to detect split reads and gapped alignments, which are then clustered to define putative SVs. Next, SAVANA applies a machine learning-informed set of heuristics to remove false positives arising from mapping errors and sequencing artifacts. Extensively validated against a multi-platform truthset, we show that SAVANA identifies a range of somatic rearrangements with high recall and precision, outperforming existing tools while maintaining a lower execution time than competing methods. In patient samples, SAVANA identifies clinically relevant alterations, such as oncogenic gene fusions, with high accuracy. Additionally, SAVANA permits the reconstruction of double minutes, multi-chromosomal chromothripsis events, and SVs mapping to highly repetitive regions, including centromeres. In sum, SAVANA permits the characterization of complex structural variants and can uncover clinically relevant mutations across diverse cancer types with high accuracy. Citation Format: Hillary Elrick, Jose Espejo Valle-Inclan, Katherine E. Trevers, Francesc Muyas, Rita Cascão, Angela Afonso, Cláudia C. Faria, Adrienne M. Flanagan, Isidro Cortés-Ciriano. SAVANA: a computational method to characterize structural variation in human cancer genomes using nanopore sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB080.