Extensive Deletions Research Articles

Genetic Variants in KNDy Pathway Lack Association with Premature Ovarian Insufficiency in Mexican Women: A Sequencing-Based Cohort Study.

Previous studies have demonstrated the essential role of the Kisspeptin/Neurokinin B/Dynorphin A (KNDy) pathway in female reproductive biology by regulating the activity of the hypothalamic-pituitary-gonadal axis. Identified loss-of-function mutations in these genes are linked to various reproductive disorders. This study investigated genetic disorders linked to mutations in the KNDy genes related to premature ovarian insufficiency (POI). A cohort of 14 Mexican POI patients underwent genetic screening using PCR-SSCP and Sanger sequencing, assessing the genetic variations' impact on protein function thereafter using multiple in silico tools. The PCR excluded extensive deletions, insertions, and duplications, while SSCP detected five genetic variants. Variations occurred in the KISS1 (c.58G>A and c.242C>G), KISS1R (c.1091A>T), PDYN (c.600C>T), and OPRK1 (c.36G>T) genes, whereas no genetic anomalies were found in NK3/NK3R genes. Each single-nucleotide variant underwent genotyping using PCR-SSCP in 100 POI-free subjects. Their allelic frequencies paralleled the patient group. These observations indicate that allelic variations in the KNDy genes may not contribute to POI etiology. Hence, screening for mutations in KNDy genes should not be a part of the diagnostic protocol for POI.

A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius

Parageobacillus thermoglucosidasius is emerging as a highly promising thermophilic organism for metabolic engineering. The utilization of CRISPR-Cas technologies has facilitated programmable genetic manipulation in P. thermoglucosidasius. However, the absence of thermostable NHEJ enzymes limited the capability of the endogenous type I CRISPR-Cas system to generate a variety of extensive genomic deletions. Here, two thermophilic NHEJ enzymes were identified and combined with the endogenous type I CRISPR-Cas system to develop a genetic manipulation tool that can achieve long-range genomic deletion across various lengths. By optimizing this tool—through adjusting the expression level of NHEJ enzymes and leveraging our discovery of a negative correlation between GC content of the guide RNA (gRNA) and deletion efficacy—we streamlined a comprehensive gRNA selection manual for whole-genome editing, achieving a 100 % success rate in randomly selecting gRNAs. Notably, using just one gRNA, we achieved genomic deletions spanning diverse length, exceeding 200 kilobases. This tool will facilitate the genomic manipulation of P. thermoglucosidasius for both fundamental research and applied engineering studies, further unlocking its potential as a thermophilic cell factory.

Identification of Essential Components of RNA Binding Domain of TLS/FUS

TLS/FUS is RNA-binding protein having multiple functions of regulations of genes, homeostasis, and cellular growth. Recent studies show that TLS is involved in phase separation and occasionally forms precipitation related to neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). RNA has been reported to suppress phase separation, droplet formation, and concomitant precipitation of TLS, suggesting that RNA is a possible candidate for ALS drug discovery. Our experiments demonstrated that a long noncoding RNA, promoter-associated noncoding RNA (pncRNA-D), specifically binds TLS and represses its phase separation and precipitation. To obtain competent drug seeds, it is essential to reveal mechanism of action of lncRNAs with specificity to TLS and inhibitory activity on phase separation and related precipitation. For this purpose, several lncRNAs (lncRNAs 1 to 6) were selected upon assays with GST-TLS binding and inhibition on the precipitation. With criteria of binding specificity for TLS, lncRNA3 has been selected for further analysis for RNA-binding ability. Initially, RNA-binding region at TLS amino acid sequence was identified from four fragments of TLS. RNA binding assay with biotinylated lncRNA3 precipitated with avidin magnetic beads indicated clearly that TLS binds the fragment 4 (373-526 aa), C-terminus end of TLS. Then, dissecting fragment 4 presents four regions, RGG2, zinc finger, RGG3, and the nuclear localization signal (NLS) region in this order. Experiments with extensive deletion mutants indicated that just one deletion out of the four regions irs not enough to delete the TLS binding, although combinatorial deletion of zinc finger with other three regions almost wiped off the lncRNA3 binding. Remarkably, each of four regions alone has no binding to TLS, either. Collectively, RGG2, zinc finger, RGG3, and NLS all are essential for binding to lncRNA3, but are required to work synergistically for full binding. These data indicate that dynamic assembly of RNA-binding domain works for action of lncRNAs and possibly has allosteric effect on intrinsically disordered region (IDR) of N-terminus of TLS, implying relation of RNA-binding with phase separation and the resultant precipitation.

RNA editing catalytic complexes edit multiple mRNA sites non-processively in Trypanosoma brucei

RNA editing generates mature mitochondrial mRNAs in T. brucei by extensive uridine insertion and deletion at numerous editing sites (ESs) as specified by guide RNAs (gRNAs). The editing is performed by three RNA Editing Catalytic Complexes (RECCs) which each have a different endonuclease in addition to 12 proteins in common resulting in RECC1 that is specific for deletion ESs and RECC2 and RECC3 that are specific for insertion ESs. Thus, different RECCs are required for editing of mRNA sequence regions where single gRNAs specify a combination of insertion and deletion ESs. We investigated how the three different RECCs might edit combinations of insertion and deletion ESs that are specified by single gRNAs by testing whether their endonuclease compositions are stable or dynamic during editing. We analyzed in vivo BirA* proximity labeling and found that the endonucleases remain associated with their set of common RECC proteins during editing when expressed at normal physiological levels. We also found that overexpression of endonuclease components resulted in minor effects on RECCs but did not affect growth. Thus, the protein stoichiometries that exist within each RECC can be altered by perturbations of RECC expression levels. These results indicate that editing of consecutive insertion and deletion ESs occurs by successive engagement and disengagement of RECCs, i.e., is non-processive, which is likely the case for consecutive pairs of insertion or deletion ESs. This clarifies the nature of the complex patterns of partially edited mRNAs that occur in vivo.

Abstract A014: Modeling recurrent chromosomal alterations in renal cell carcinoma evolution

Abstract The loss of one copy of chromosome 3p represents an early truncal genetic event in the majority of clear cell renal cell carcinoma (ccRCC) tumors. Chromosome 3p loss can be initiated by chromothripsis, a process in which mis-segregated chromosomes entrapped within abnormal nuclear structures called micronuclei become pulverized into small genomic fragments. These fragments are then re-stitched together to form complex rearrangements that are accompanied by extensive segmental deletions that inactivate critical tumor suppressor genes located on chromosome 3p. Despite being a driver of ccRCC development, the mechanism(s) that initiate chromosome 3p chromothripsis followed by pressures that select for specific rearrangement patterns are poorly understood. To recapitulate early genetic events arising throughout ccRCC evolution, here we developed chromosome 3p-specific micronuclei models in non-transformed human renal proximal tubule epithelial cells (RPTECs) to interrogate recurrent patterns of chromosomal alterations that promote renal cell tumorigenesis. To do so, we used CRISPR/Cas9 to induce a DNA double-strand break on chromosome 3p, which generates chromosome 3p-specific micronuclei when the acentric arm is left unrepaired into the subsequent mitosis. DNA fluorescence in situ hybridization revealed that mis-segregation of chromosome 3p into micronuclei induces chromosome pulverization and rearrangements. Notably, these alterations are sufficient to provide a selective advantage by enabling anchorage-independent growth in vitro. Cytogenetic characterization of partially transformed RPTECs revealed enrichment of chromosome 3p rearrangements, which were gradually lost upon propagation as conventional monolayer cultures. These results provide mechanistic insight into the origins of chromosome 3p alterations in ccRCC and further highlight the role of selection pressure in shaping genome evolution. Citation Format: Rashmi Dahiya, Peter Ly. Modeling recurrent chromosomal alterations in renal cell carcinoma evolution [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A014.

Mycoplasma DnaK increases DNA copy number variants in vivo

The human microbiota affects critical cellular functions, although the responsible mechanism(s) is still poorly understood. In this regard, we previously showed that Mycoplasma fermentans DnaK, an HSP70 chaperone protein, hampers the activity of important cellular proteins responsible for DNA integrity. Here, we describe a novel DnaK knock-in mouse model generated in our laboratory to study the effect of M. fermentans DnaK expression in vivo. By using an array-based comparative genomic hybridization assay, we demonstrate that exposure to DnaK was associated with a higher number of DNA copy number variants (CNVs) indicative of unbalanced chromosomal alterations, together with reduced fertility and a high rate of fetal abnormalities. Consistent with their implication in genetic disorders, one of these CNVs caused a homozygous Grid2 deletion, resulting in an aberrant ataxic phenotype that recapitulates the extensive biallelic deletion in the Grid2 gene classified in humans as autosomal recessive spinocerebellar ataxia 18. Our data highlight a connection between components of the human urogenital tract microbiota, namely Mycoplasmas, and genetic abnormalities in the form of DNA CNVs, with obvious relevant medical, diagnostic, and therapeutic implications.

Complete genomic characterization of bovine papillomavirus type 1 and 2 strains infers ongoing cross-species transmission between cattle and horses

Infection with bovine papillomavirus (BPV) types 1 and 2 results in the most common skin tumor of horses, termed equine sarcoid. The persistent and recurrent nature of this tumor stands in contrast to the regressive nature of BPV-1/− 2 induced cutaneous papillomas in cattle. The circulation of horse-specific BPV-1/− 2 variants within equine populations has been suggested as a possible explanation for the difference in clinical presentation of BPV-1/− 2 infection between horses and cattle. In order to investigate this hypothesis, we identified 98 complete BPV-1/− 2 genomes using a Nanopore sequencing approach. Separate BPV-1/− 2 alignments were used to infer Bayesian phylogenetic trees. Phylogeny-trait association concerning host species was investigated using Bayesian Tip-association Significance software (BaTS) Overall, 179 unique BPV-1 and 128 BPV-2 substitutions were found. The E2 coding region in the viral genome exhibited an exceptionally high rate of non-synonymous mutations (81 %, n = 13/16). Interestingly, extensive deletions in the L1/L2 region (up to 1.5 kb) were found exclusively in horse-derived samples. Nevertheless, the most frequently detected single nucleotide polymorphisms were shared between equine and bovine hosts, which is in agreement with BaTS results indicating no phylogeny-host correlation. We found indications that horse-specific mutations might exist in subpopulations of equine derived BPV-1/− 2, but these did not result in horse-adapted genetic variants. Based on these observations, cross-species transmission from cattle to horses seems to be an ongoing process, rather than an ancient occurrence that has been followed by the circulation of horse-adapted BPV variants in the horse population..

Unanticipated Large-Scale Deletion in Fusarium graminearum Genome Using CRISPR/Cas9 and Its Impact on Growth and Virulence

Fusarium graminearum, a filamentous fungus, and causal agent of Fusarium head blight (FHB) in wheat and other cereals, leads to significant economic losses globally. This study aimed to investigate the roles of specific genes in F. graminearum virulence using CRISPR/Cas9-mediated gene deletions. Illumina sequencing was used to characterize the genomic changes due to editing. Unexpectedly, a large-scale deletion of 525,223 base pairs on chromosome 2, comprising over 222 genes, occurred in two isolates. Many of the deleted genes were predicted to be involved in essential molecular functions, such as oxidoreductase activity, transmembrane transporter activity, hydrolase activity, as well as biological processes, such as carbohydrate metabolism and transmembrane transport. Despite the substantial loss of genetic material, the mutant isolate exhibited normal growth rates and virulence on wheat under most conditions. However, growth rates were significantly reduced under high temperatures and on some media. Additionally, wheat inoculation assays using clip dipping, seed inoculation, and head point inoculation methods were performed. No significant differences in virulence were observed, suggesting that these genes were not involved in infection or alternative compensatory pathways, and allow the fungi to maintain pathogenicity despite the extensive genomic deletion.

The genome of okra (Abelmoschus esculentus) provides insights into its genome evolution and high nutrient content.

Okra (Abelmoschus esculentus) is an important vegetable crop with high nutritional value. However, the mechanism underlying its high nutrient content remains poorly understood. Here, we present a chromosome-scale genome of okra with a size of 1.19Gb. Comparative genomics analysis revealed the phylogenetic status of A. esculentus, as well as whole-genome duplication (WGD) events that have occurred widely across the Malvaceae species. We found that okra has experienced three additional WGDs compared with the diploid cotton Gossypium raimondii, resulting in a large chromosome number (2n = 130). After three WGDs, okra has undergone extensive genomic deletions and retained substantial numbers of genes related to secondary metabolite biosynthesis and environmental adaptation, resulting in significant differences between okra and G. raimondii in the gene families related to cellulose synthesis. Combining transcriptomic and metabolomic analysis, we revealed the relationship between gene expression and metabolite content change across different okra developmental stages. Furthermore, the sinapic acid/S-lignin biosynthesis-related gene families have experienced remarkable expansion in okra, and the expression of key enzymes involved in the sinapic acid/S-lignin biosynthesis pathway vary greatly across developmental periods, which partially explains the differences in metabolite content across the different stages. Our study gains insights into the comprehensive evolutionary history of Malvaceae species and the genetic basis that underlies the nutrient content changes in okra, which will facilitate the functional study and genetic improvement of okra varieties.

Novel genetically engineered mouse models for clear cell renal cell carcinoma

Genetically engineered mouse models (GEMMs) are important immunocompetent models for research into the roles of individual genes in cancer and the development of novel therapies. Here we use inducible CRISPR-Cas9 systems to develop two GEMMs which aim to model the extensive chromosome p3 deletion frequently observed in clear cell renal cell carcinoma (ccRCC). We cloned paired guide RNAs targeting early exons of Bap1, Pbrm1, and Setd2 in a construct containing a Cas9D10A (nickase, hSpCsn1n) driven by tetracycline (tet)-responsive elements (TRE3G) to develop our first GEMM. The founder mouse was crossed with two previously established transgenic lines, one carrying the tet-transactivator (tTA, Tet-Off) and one with a triple-mutant stabilized HIF1A-M3 (TRAnsgenic Cancer of the Kidney, TRACK), both driven by a truncated, proximal tubule-specific γ-glutamyltransferase 1 (ggt or γGT) promoter, to create triple-transgenic animals. Our results indicate that this model (BPS-TA) induces low numbers of somatic mutations in Bap1 and Pbrm1 (but not in Setd2), known tumor suppressor genes in human ccRCC. These mutations, largely restricted to kidneys and testis, induced no detectable tissue transformation in a cohort of 13 month old mice (N = 10). To gain insights into the low frequencies of insertions and deletions (indels) in BPS-TA mice we analyzed wild type (WT, N = 7) and BPS-TA (N = 4) kidneys by RNAseq. This showed activation of both DNA damage and immune response, suggesting activation of tumor suppressive mechanisms in response to genome editing. We then modified our approach by generating a second model in which a ggt-driven, cre-regulated Cas9WT(hSpCsn1) was employed to introduce Bap1, Pbrm1, and Setd2 genome edits in the TRACK line (BPS-Cre). The BPS-TA and BPS-Cre lines are both tightly controlled in a spatiotemporal manner with doxycycline (dox) and tamoxifen (tam), respectively. In addition, whereas the BPS-TA line relies on paired guide RNAs (gRNAs), the BPS-Cre line requires only single gRNAs for gene perturbation. In the BPS-Cre we identified increased Pbrm1 gene-editing frequencies compared to the BPS-TA model. Whereas we did not detect Setd2 edits in the BPS-TA kidneys, we found extensive editing of Setd2 in the BPS-Cre model. Bap1 editing efficiencies were comparable between the two models. Although no gross malignancies were observed in our study, this is the first reported GEMM which models the extensive chromosome 3p deletion frequently observed in kidney cancer patients. Further studies are required (1) to model more extensive 3p deletions, e.g. impacting additional genes, and (2) to increase the cellular resolution, e.g. by employing single-cell RNAseq to ascertain the effects of specific combinatorial gene inactivation.

Spike-Independent Infection of Human Coronavirus 229E in Bat Cells.

Bats are the reservoir for numerous human pathogens, including coronaviruses. Despite many coronaviruses having descended from bat ancestors, little is known about virus-host interactions and broader evolutionary history involving bats. Studies have largely focused on the zoonotic potential of coronaviruses with few infection experiments conducted in bat cells. To determine genetic changes derived from replication in bat cells and possibly identify potential novel evolutionary pathways for zoonotic virus emergence, we serially passaged six human 229E isolates in a newly established Rhinolophus lepidus (horseshoe bat) kidney cell line. Here, we observed extensive deletions within the spike and open reading frame 4 (ORF4) genes of five 229E viruses after passaging in bat cells. As a result, spike protein expression and infectivity of human cells was lost in 5 of 6 viruses, but the capability to infect bat cells was maintained. Only viruses that expressed the spike protein could be neutralized by 229E spike-specific antibodies in human cells, whereas there was no neutralizing effect on viruses that did not express the spike protein inoculated on bat cells. However, one isolate acquired an early stop codon, abrogating spike expression but maintaining infection in bat cells. After passaging this isolate in human cells, spike expression was restored due to acquisition of nucleotide insertions among virus subpopulations. Spike-independent infection of human coronavirus 229E may provide an alternative mechanism for viral maintenance in bats that does not rely on the compatibility of viral surface proteins and known cellular entry receptors. IMPORTANCE Many viruses, including coronaviruses, originated from bats. Yet, we know little about how these viruses switch between hosts and enter human populations. Coronaviruses have succeeded in establishing in humans at least five times, including endemic coronaviruses and the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In an approach to identify requirements for host switches, we established a bat cell line and adapted human coronavirus 229E viruses by serial passage. The resulting viruses lost their spike protein but maintained the ability to infect bat cells, but not human cells. Maintenance of 229E viruses in bat cells appears to be independent of a canonical spike receptor match, which in turn might facilitate cross-species transmission in bats.

Genome Protection by DNA Polymerase θ.

DNA polymerase θ (Pol θ) is a DNA repair enzyme widely conserved in animals and plants. Pol θ uses short DNA sequence homologies to initiate repair of double-strand breaks by theta-mediated end joining. The DNA polymerase domain of Pol θ is at the C terminus and is connected to an N-terminal DNA helicase-like domain by a central linker. Pol θ is crucial for maintenance of damaged genomes during development, protects DNA against extensive deletions, and limits loss of heterozygosity. The cost of using Pol θ for genome protection is that a few nucleotides are usually deleted or added at the repair site. Inactivation of Pol θ often enhances the sensitivity of cells to DNA strand-breaking chemicals and radiation. Since some homologous recombination-defective cancers depend on Pol θ for growth, inhibitors of Pol θ may be useful in treating such tumors.

Mitochondrial RNA editing in Trypanoplasma borreli: New tools, new revelations

The kinetoplastids are unicellular flagellates that derive their name from the ‘kinetoplast’, a region within their single mitochondrion harboring its organellar genome of high DNA content, called kinetoplast (k) DNA. Some protein products of this mitochondrial genome are encoded as cryptogenes; their transcripts require editing to generate an open reading frame. This happens through RNA editing, whereby small regulatory guide (g)RNAs direct the proper insertion and deletion of one or more uridines at each editing site within specific transcript regions. An accurate perspective of the kDNA expansion and evolution of their unique uridine insertion/deletion editing across kinetoplastids has been difficult to achieve. Here, we resolved the kDNA structure and editing patterns in the early-branching kinetoplastid Trypanoplasma borreli and compare them with those of the well-studied trypanosomatids. We find that its kDNA consists of circular molecules of about 42 kb that harbor the rRNA and protein-coding genes, and 17 different contigs of approximately 70 kb carrying an average of 23 putative gRNA loci per contig. These contigs may be linear molecules, as they contain repetitive termini. Our analysis uncovered a putative gRNA population with unique length and sequence parameters that is massive relative to the editing needs of this parasite. We validated or determined the sequence identity of four edited mRNAs, including one coding for ATP synthase 6 that was previously thought to be missing. We utilized computational methods to show that the T. borreli transcriptome includes a substantial number of transcripts with inconsistent editing patterns, apparently products of non-canonical editing. This species utilizes the most extensive uridine deletion compared to other studied kinetoplastids to enforce amino acid conservation of cryptogene products, although insertions still remain more frequent. Finally, in three tested mitochondrial transcriptomes of kinetoplastids, uridine deletions are more common in the raw mitochondrial reads than aligned to the fully edited, translationally competent mRNAs. We conclude that the organization of kDNA across known kinetoplastids represents variations on partitioned coding and repetitive regions of circular molecules encoding mRNAs and rRNAs, while gRNA loci are positioned on a highly unstable population of molecules that differ in relative abundance across strains. Likewise, while all kinetoplastids possess conserved machinery performing RNA editing of the uridine insertion/deletion type, its output parameters are species-specific.

Phylostat: a web-based tool to analyze paralogous clade divergence in phylogenetic trees

Phylogenetic trees are useful tools to infer evolutionary relationships between genetic entities. Phylogenetics enables not only evolution-based gene clustering but also the assignment of gene duplication and deletion events to the nodes when coupled with statistical approaches such as bootstrapping. However, extensive gene duplication and deletion events bring along a challenge in interpreting phylogenetic trees and require manual inference. In particular, there has been no robust method of determining whether one of the paralog clades systematically shows higher divergence following the gene duplication event as a sign of functional divergence. Here, we provide Phylostat, a graphical user interface that enables clade divergence analysis, visually and statistically. Phylostat is a web-based tool built on phylo.io to allow comparative clade divergence analysis, which is available at https://phylostat.adebalilab.org under an MIT open-source licence.

Consonants lost: a Swedish girl with protracted phonological development

ABSTRACT This paper addresses the phonology of a Swedish girl, aged 3 years 10 months, with extensive phonological difficulties that include an unusual phonological pattern. She had relatively well-developed phonological building blocks in terms of features, stress pattern and word length (number of syllables), but had extensive difficulties regarding syllable and word shapes, with frequent deletions of both segments and whole syllables. Word-initial position was dominated by non-continuant consonants, both voiced and voiceless, with extensive deletion. Word-medial position was dominated by voiceless obstruents, with extensive use of [j] in place of voiced consonants. Word-final position was similar to word-medial, but with more deletion than [j]. Non-initial stressed syllables resemble word-initial syllables. Consonant clusters mirrored singletons, i.e. with similar substitution patterns or deletions. We present a non-linear analysis of her speech production, and a plan for intervention targeting her special difficulties.

An Oil Hyper-Accumulator Mutant Highlights Peroxisomal ATP Import as a Regulatory Step for Fatty Acid Metabolism in Aurantiochytrium limacinum.

Thraustochytrids are marine protists that naturally accumulate triacylglycerol with long chains of polyunsaturated fatty acids, such as ω3-docosahexaenoic acid (DHA). They represent a sustainable response to the increasing demand for these “essential” fatty acids (FAs). Following an attempt to transform a strain of Aurantiochytrium limacinum, we serendipitously isolated a clone that did not incorporate any recombinant DNA but contained two to three times more DHA than the original strain. Metabolic analyses indicated a deficit in FA catabolism. However, whole transcriptome analysis did not show down-regulation of genes involved in FA catabolism. Genome sequencing revealed extensive DNA deletion in one allele encoding a putative peroxisomal adenylate transporter. Phylogenetic analyses and yeast complementation experiments confirmed the gene as a peroxisomal adenylate nucleotide transporter (AlANT1), homologous to yeast ScANT1 and plant peroxisomal adenylate nucleotide carrier AtPNC genes. In yeast and plants, a deletion of the peroxisomal adenylate transporter inhibits FA breakdown and induces FA accumulation, a phenotype similar to that described here. In response to this metabolic event, several compensatory mechanisms were observed. In particular, genes involved in FA biosynthesis were upregulated, also contributing to the high FA accumulation. These results support AlANT1 as a promising target for enhancing DHA production in Thraustochytrids.

A tetraploidization event shaped the Aquilaria sinensis genome and contributed to the ability of sesquiterpenes synthesis

BackgroundAgarwood, generated from the Aquilaria sinensis, has high economic and medicinal value. Although its genome has been sequenced, the ploidy of A. sinensis paleopolyploid remains unclear. Moreover, the expression changes of genes associated with agarwood formation were not analyzed either.ResultsIn the present work, we reanalyzed the genome of A. sinensis and found that it experienced a recent tetraploidization event ~ 63–71 million years ago (Mya). The results also demonstrated that the A. sinensis genome had suffered extensive gene deletion or relocation after the tetraploidization event, and exhibited accelerated evolutionary rates. At the same time, an alignment of homologous genes related to different events of polyploidization and speciation were generated as well, which provides an important comparative genomics resource for Thymelaeaceae and related families. Interestingly, the expression changes of genes related to sesquiterpene synthesis in wounded stems of A. sinensis were also observed. Further analysis demonstrated that polyploidization promotes the functional differentiation of the key genes in the sesquiterpene synthesis pathway.ConclusionsBy reanalyzing its genome, we found that the tetraploidization event shaped the A. sinensis genome and contributed to the ability of sesquiterpenes synthesis. We hope that these results will facilitate our understanding of the evolution of A. sinensis and the function of genes involved in agarwood formation.

Abstract 81: Studying Ghanian Cancer Genomes Using Cell-free DNA

Abstract Purpose: Analysis of cell free DNA could provide a rapid and non-invasive approach to detect cancer and provide new molecular insights in many African countries where expert pathology is lacking. Hence, we tested whether whole-genome sequencing of cfDNA (WGS-cfDNA) could identify somatic alterations that drive breast cancer. Methods: We conducted a pilot on 15 Ghanaian women (median age 49.5 years) recruited as part of the Ghana Breast Health Study. cfDNA was extracted and subjected to WGS at 30x and 0.1x. ichorCNA software was used to predict copy number alterations and ctDNA fractions. Results: We found extensive amplification and deletion of multiple chromosomal regions including those with oncogenes and tumor suppressor genes associated with breast cancer. Similar copy number alterations for selected breast cancer genes were observed with 0.1x and 30x cfDNA-WGS with increasing concordance between the two instruments as the ctDNA fraction increases. We observed a high frequency (>50%) of copy number gain in 3/5 regions and potential target genes for the amplification (chr8p11-12 [ZNF703] n=8, 53.3%; chr8q24.2 [MYC] n=9, 60%; chr19q12 [CCNE1] n=9, 60%), which were in agreement to previous observations among African-American (AA) ancestry compared to European-American (EA) ancestry in TCGA datasets. Conclusion: Our data provided evidence that ctDNA-based genomic studies are possible and ctDNA analysis could be a tool for future molecular oncology studies in Africa for cancer etiology, surveillance and clinical trials. Citation Format: Samuel Ahuno, Anna-Lisa Doebley, Thomas Ahearn, Joel Yarney, Nicholas Titiloye, Nancy Hamel, Ernest Adjei, Joe-Nat Clegg-Lamptey, Lawrence Edusei, Baffour Awuah, Xiaoyu Song, Verne Vanderpuye, Mustapha Abubakar, Maire Duggan, Daniel Stover, Kofi Nyarko, John Bartlet, Francis Aitpillah, Daniel Ansong, Kevin Gardner, Anne Bowcock, Carlos Caldas, William Foulkes, Seth Wiafe, Wiafe-Addai, Montserrat Garcia-Closas, Alexander Kwarteng, Gavin Ha, Jonine Figueroa, Paz Polak, On Behalf Of Ghana Breast Health Study Team. Studying Ghanian Cancer Genomes Using Cell-free DNA [abstract]. In: Proceedings of the 9th Annual Symposium on Global Cancer Research; Global Cancer Research and Control: Looking Back and Charting a Path Forward; 2021 Mar 10-11. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2021;30(7 Suppl):Abstract nr 81.

Aspergillus fumigatus, One Uninucleate Species with Disparate Offspring.

Establishment of a fungal infection due to Aspergillus fumigatus relies on the efficient germination of the airborne conidia once they penetrate the respiratory tract. However, the features of conidial germination have been poorly explored and understood in this fungal species as well as in other species of filamentous fungi. We show here that the germination of A. fumigatus is asynchronous. If the nutritional environment and extensive gene deletions can modify the germination parameters for A. fumigatus, the asynchrony is maintained in all germinative conditions tested. Even though the causes for this asynchrony of conidial germination remain unknown, asynchrony is essential for the completion of the biological cycle of this filamentous fungus.

Abstract PO-093: Circulating tumor DNA (ctDNA) from peripheral blood is detectable among Ghanaian breast cancer patients

Abstract Cancer incidence is rising and mortality rates are high in Africa, where access to molecular pathology is limited. Analysis of cancer-related mutations in circulating tumor DNA (ctDNA) from cell-free DNA (cfDNA) that is shed into the bloodstream by tumor cells could be transformative to the African continent and provide new molecular insights. Using samples collected from the Ghana Breast Health Study we tested whether whole-genome sequencing (WGS) of cfDNA could detect ctDNA and identify somatic alterations that drive breast cancer. We selected 15 breast cancer patients (median age 49.5 years) with duplicated plasma samples. Pathologic grade, age, and immunohistochemical (IHC) stains for estrogen receptor (ER), progesterone receptor (PR) and HER2 were available for the majority of patients (>80%). cfDNA extraction and WGS at 30x and 0.1x was performed. ichorCNA software was used on Next Generation Sequencing (NGS) read counts to estimate the ctDNA fraction and predict copy number alteration profiles. High depth 30x cfDNA-WGS analysis showed that all 15 breast cancer patients had 1% ctDNA or greater (median[IQR] 3.96%[2.22%-8.13%]). There was high concordance between estimated ctDNA fraction using 0.1x and 30x WGS (Pearson r = 0.9). Copy number profiling showed extensive amplification and deletion of multiple chromosomal regions containing important cancer genes (such as MYC, PIK3CA, TERT, and GATA3). Of the four patients classified as HER2 positive based on IHC, two had increased ERBB2 copy number (50 and 3 copies, respectively). Our data provide evidence that ctDNA-based genomic studies are possible and ctDNA analysis could be a tool for future molecular oncology studies in Africa for cancer etiology, surveillance and clinical trials. Citation Format: Samuel T. Ahuno, Lawrence Edusei, Nicolas Titiloye, Ernest Adjei, Joe-Nat Clegg-Lamptey, Joel Yarney, Beatrice Wiafe-Addai, Baffour Awuah, Verne Vanderpuye, Maire Duggan, Seth Wiafe, Kofi Nyarko, Francis Aitpillah, Daniel Ansong, Thomas Ahearn, Alexander Kwarteng, Mustapha Abubakar, Montserrat Garcia-Closas, Gavin Ha, Jonine D. Figueroa, Paz Polak, on behalf of the Ghana Breast Health Study Team. Circulating tumor DNA (ctDNA) from peripheral blood is detectable among Ghanaian breast cancer patients [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-093.