Levels Of Copy Number Variation Research Articles

Abstract 2262: Genomic profiling of leukemic cell lines, responders vs. non-responders to Narazaciclib (HX301 or ON123300), a novel kinase inhibitor with activity against CSF1R and FLT3

Abstract Acute myeloid leukemia (AML) is an aggressive myeloid leukemia with global annual mortality ~150,000. While standard of care (SOC) of the 1st -line treatment being mainly induction chemotherapy, available targeted therapies cover rather limited patients carrying specific driver mutations (IDH mutations and FLT3-ITR). We previously reported a novel kinase inhibitor, Narazaciclib with strong anti-CSF1R activity (IC50 0.7nM) as well as anti-FLT3 (IC50 7nM), and also thus anti-AML activity for certain AML experimental models, as shown in vitro and in vivo (AACR Annual Meeeting, 2023). In the present study, we assessed the impact of HX301 on the in vitro proliferations of a panel of leukemic cell lines (n = 54), including a panel of AML cell lines (n = 33), that have been parallelly genomic-profiled by whole-exome sequencing and whole-transcriptome sequencing. We then set out to analyze the proliferation data and the genomic data, in attempt to elucidate genes and pathways that may be associated with the treatment responses. The biomarker analysis has been performed on gene expression, mutation and copy number variation levels, and the data been presented using common methodologies, including principal component analysis, differential expression analysis, correlation analysis, Gene Ontology enrichment analysis, gene set variation analysis, somatic gene mutation analysis, and copy number variation analysis, etc. Some of the observations have been made: 1) differential expression analysis on all leukemic cell lines shows that 254 genes have been positively associated with the responders; while expression of 191 genes are negatively correlated with AUC, i.e. positively associated with response, as revealed by Spearman correlation analysis; 2) Gene Ontology enrichment analysis revealed these response related genes are mostly involved in myeloid leukocyte activation and defense response; 3) all FLT3-ITD AML lines are responders, presumably due to the FLT3i activity of HX301; 4) CSF1R expression levels seem not correlated to drug responses; 5) pathway analysis on non FLT3-ITD AML lines reveals that STAT5 activation downstream of FLT3 signaling, IL7 pathway and NFKB pathway are significantly associated with better response. Finally, we are also attempted to use statistics learning to reveal genes, as a potential predictive biomarker, that may be correlated to drug response. Citation Format: Jia Xue, Tao Yang, Hang Ke, Sheng Guos, Henry Qixiang Li. Genomic profiling of leukemic cell lines, responders vs. non-responders to Narazaciclib (HX301 or ON123300), a novel kinase inhibitor with activity against CSF1R and FLT3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2262.

Elucidating the role of tumor-associated ALOX5+ mast cells with transformative function in cervical cancer progression via single-cell RNA sequencing.

Cervical cancer (CC) is the fourth most common malignancy among women globally and serves as the main cause of cancer-related deaths among women in developing countries. The early symptoms of CC are often not apparent, with diagnoses typically made at advanced stages, which lead to poor clinical prognoses. In recent years, numerous studies have shown that there is a close relationship between mast cells (MCs) and tumor development. However, research on the role MCs played in CC is still very limited at that time. Thus, the study conducted a single-cell multi-omics analysis on human CC cells, aiming to explore the mechanisms by which MCs interact with the tumor microenvironment in CC. The goal was to provide a scientific basis for the prevention, diagnosis, and treatment of CC, with the hope of improving patients' prognoses and quality of life. The present study acquired single-cell RNA sequencing data from ten CC tumor samples in the ArrayExpress database. Slingshot and AUCcell were utilized to infer and assess the differentiation trajectory and cell plasticity of MCs subpopulations. Differential expression analysis of MCs subpopulations in CC was performed, employing Gene Ontology, gene set enrichment analysis, and gene set variation analysis. CellChat software package was applied to predict cell communication between MCs subpopulations and CC cells. Cellular functional experiments validated the functionality of TNFRSF12A in HeLa and Caski cell lines. Additionally, a risk scoring model was constructed to evaluate the differences in clinical features, prognosis, immune infiltration, immune checkpoint, and functional enrichment across various risk scores. Copy number variation levels were computed using inference of copy number variations. The obtained 93,524 high-quality cells were classified into ten cell types, including T_NK cells, endothelial cells, fibroblasts, smooth muscle cells, epithelial cells, B cells, plasma cells, MCs, neutrophils, and myeloid cells. Furthermore, a total of 1,392 MCs were subdivided into seven subpopulations: C0 CTSG+ MCs, C1 CALR+ MCs, C2 ALOX5+ MCs, C3 ANXA2+ MCs, C4 MGP+ MCs, C5 IL32+ MCs, and C6 ADGRL4+ MCs. Notably, the C2 subpopulation showed close associations with tumor-related MCs, with Slingshot results indicating that C2 subpopulation resided at the intermediate-to-late stage of differentiation, potentially representing a crucial transition point in the benign-to-malignant transformation of CC. CNVscore and bulk analysis results further confirmed the transforming state of the C2 subpopulation. CellChat analysis revealed TNFRSF12A as a key receptor involved in the actions of C2 ALOX5+ MCs. Moreover, in vitro experiments indicated that downregulating the TNFRSF12A gene may partially inhibit the development of CC. Additionally, a prognosis model and immune infiltration analysis based on the marker genes of the C2 subpopulation provided valuable guidance for patient prognosis and clinical intervention strategies. We first identified the transformative tumor-associated MCs subpopulation C2 ALOX5+ MCs within CC, which was at a critical stage of tumor differentiation and impacted the progression of CC. In vitro experiments confirmed the inhibitory effect of knocking down the TNFRSF12A gene on the development of CC. The prognostic model constructed based on the C2 ALOX5+MCs subset demonstrated excellent predictive value. These findings offer a fresh perspective for clinical decision-making in CC.

Molecular and cellular aspects of the impact of secondary metabolites from common barberry and hybrid goat’s rue on the HeLa cell line

Purpose of the study. To isolate and verify pure fractions of secondary plant metabolites contained in B. vulgaris (L.) and P. hibridus (L.), as well as to conduct a model experiment and molecular genetic study to evaluate their cytotoxic effect in vitro on the HeLa cell line.Materials and methods. The isolation and verification of all compounds used in the experiment were carried out using column chromatography and nuclear magnetic resonance methods at the Department of Natural Compounds, Faculty of Chemistry, Southern Federal University. Subsequently, an experiment was conducted using cultural and molecular methods on the HeLa cell line in three repetitions for each test compound; after incubation with them, the numbers of dead cells were counted on the automated NanoEnTek JuliFl counter, and the numbers of cells in apoptosis were measured by flow cytometry on the BD analyzer FACS Canto II. The level of copy number variation and expression of genes responsible for apoptosis were assessed by real-time PCR (RT-PCR). In total, three substances were studied, with two concentrations (4 and 12 μg/ml) and two exposures (24 and 72 hours) for each of them.Results. In the first stage of the study, we isolated and verified the berberine alkaloid extracted from the roots of B. vulgaris (L.), as well as 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one and 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde from P. hibridus (L.). The subsequent stage of the study demonstrated the maximal cellular death under the action of berberine at a 72-hour exposure. However, the RT-PCR assessment of the copy number variation and expression of the CASP8, CASP9, CASP3, BAX, BCL2, TP53 and MDM2 genes revealed the presence of apoptosis initiation in tumor cells at the molecular level under the action of all the studied compounds: both berberine and furan and azulene derivatives derived from P. hibridus (L.).Conclusion. All compounds used in the experiment exhibited a cytotoxic effect on the HeLa cell line. Berberine alkaloid showed the most pronounced cytotoxic effect on the HeLa line as recorded by all methods used in the study. Terpenoids 4-dihydroxy-2,5-dimethylfuran-3(2H)-one and 2,2,8-trimethyldecahydroazulene-5,6-dicarbaldehyde, when exposed to the HeLa line, caused an increase in the copy number variation and expression of the CASP9, CASP3 loci, which are among the main activators of apoptosis. They also influenced the expression of TP53 and MDM2 loci.

Relation between HLA and copy number variation of steroid 21-hydroxylase in a Swedish cohort of patients with autoimmune Addison's disease.

Autoantibodies against the adrenal enzyme 21-hydroxylase is a hallmark manifestation in autoimmune Addison's disease (AAD). Steroid 21-hydroxylase is encoded by CYP21A2, which is located in the HLA region together with the highly similar pseudogene CYP21A1P. A high level of copy number variation is seen for the two genes, and therefore we asked if genetic variation of the CYP21 genes is associated with AAD. Using next-generation DNA sequencing, we estimated the copy number of CYP21A2 and CYP21A1P together with HLA alleles in 479 Swedish patients with AAD and autoantibodies against 21-hydroxylase, and in 1,393 healthy controls. With 95% of individuals carrying two functional 21-hydroxylase genes, no difference in CYP21A2 copy number was found when comparing patients and controls. In contrast, we discovered a lower copy number of the pseudogene CYP21A1P among AAD patients (p = 5 x 10-44) together with associations of additional nucleotide variants in the CYP21 region. However, the strongest association was found for HLA-DQB1*02:01 (p = 9 x 10-63), which in combination with the DRB1*04:04-DQB1*03:02 haplotype imposed the greatest risk of AAD. We identified strong associations between copy number variants in the CYP21 region and risk of AAD, although these associations most likely are due to linkage disequilibrium with disease-associated HLA class II alleles.

High Levels of Copy Number Variation of Ampliconic Genes across Major Human Y Haplogroups.

Because of its highly repetitive nature, the human male-specific Y chromosome remains understudied. It is important to investigate variation on the Y chromosome to understand its evolution and contribution to phenotypic variation, including infertility. Approximately 20% of the human Y chromosome consists of ampliconic regions which include nine multi-copy gene families. These gene families are expressed exclusively in testes and usually implicated in spermatogenesis. Here, to gain a better understanding of the role of the Y chromosome in human evolution and in determining sexually dimorphic traits, we studied ampliconic gene copy number variation in 100 males representing ten major Y haplogroups world-wide. Copy number was estimated with droplet digital PCR. In contrast to low nucleotide diversity observed on the Y in previous studies, here we show that ampliconic gene copy number diversity is very high. A total of 98 copy-number-based haplotypes were observed among 100 individuals, and haplotypes were sometimes shared by males from very different haplogroups, suggesting homoplasies. The resulting haplotypes did not cluster according to major Y haplogroups. Overall, only two gene families (RBMY and TSPY) showed significant differences in copy number among major Y haplogroups, and the haplogroup of a male could not be predicted based on his ampliconic gene copy numbers. Finally, we did not find significant correlations either between copy number variation and individual’s height, or between the former and facial masculinity/femininity. Our results suggest rapid evolution of ampliconic gene copy numbers on the human Y, and we discuss its causes.

Abstract 2411: Evolution during propagation and treatment of patient-derived triple negative breast cancer xenografts

Abstract Individual tumors, including the aggressive and difficult to treat triple-negative (ER-/PR-/HER2-) breast cancers (TNBCs) are heterogeneous collections of cells with multiple subclonal populations each contributing to the tumor. While subclonal heterogeneity is likely responsible for the development of drug resistance, identification of how tumor cell populations change over time has been difficult, largely because of the challenges in resampling tumor tissue at close time points. Here we quantify tumor evolution in human patient-derived xenografts implanted into NSG mice, which we use to test subclonal heterogeneity as a function of location within a tumor, propagation time, and drug treatment. We used high-depth (∼400x) sequencing of a targeted panel of 358 genes to quantify somatic mutation allele frequencies from 6 spatially-separated and 8 temporally-propagated xenograft samples derived from the same TNBC patient tumors. Samples ranged in age from 2-4 months post-engraftment. Although we observed a few low frequency mutations distinguishing samples, overall we found that allele frequencies of somatic mutations were well-preserved on this time scale. We then generated replicate xenografts from the same patient tumor and treated them respectively with cisplatin, doxorubicin, cyclophosphamide, docetaxel, or vehicle control for 25 days. Although again somatic mutations showed few differences in allele frequency across samples, substantial variations were seen when data were analyzed for copy number alterations. To confirm these effects we repeated the treatments for xenografts derived from two additional TNBC patients. Again we observed strong changes in tumor heterogeneity at the copy number level. This effect was particularly, but not exclusively, apparent in tumors with the greatest response to therapy. We further verified these measurements through Sanger and digital PCR sequencing on the treated mice and other mice in the same cohorts. Using a multi-sample xenograft propagation, dissection, sequencing, and computational analysis protocol, we have shown that tumor subpopulation changes in response to treatment can be quantified and distinguished from spatial or temporal effects, even for treatment time courses as short as 1 month. In triple negative breast cancer these variations are most apparent at the level of copy number variation. Our study demonstrates how patient-derived xenografts can provide detailed resolution of tumor population evolution during the manifestation of resistance. Citation Format: Hyunsoo Kim, Pooja Kumar, Francesca Menghi, Joshy George, Guru Ananda, Susan Mockus, Chengsheng Zhang, Nicholas Larson, Henry C. Chen, Yan Yang, James Keck, R. Krishnamurthy Karuturi, Charles Lee, Carol Bult, Edison Liu, Jeffrey H. Chuang. Evolution during propagation and treatment of patient-derived triple negative breast cancer xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2411.

Chromosome Copy Number Variation and Control in the Ciliate Chilodonella uncinata

Copy number variations are widespread in eukaryotes. The unusual genome architecture of ciliates, in particular, with its process of amitosis in macronuclear division, provides a valuable model in which to study copy number variation. The current model of amitosis envisions stochastic distribution of macronuclear chromosomes during asexual reproduction. This suggests that amitosis is likely to result in high levels of copy number variation in ciliates, as dividing daughter cells can have variable copy numbers of chromosomes if chromosomal distribution during amitosis is a stochastic process. We examined chromosomal distribution during amitosis in Chilodonella uncinata, a ciliate with gene-size macronuclear chromosomes. We quantified 4 chromosomes in evolving populations of C. uncinata and modeled the amitotic distribution process. We found that macronuclear chromosomes differ in copy number from one another but that copy number does not change as expected under a stochastic process. The chromosome carrying SSU increased in copy number, which is consistent with selection to increase abundance; however, two other studied chromosomes displayed much lower than expected among-line variance. Our models suggest that balancing selection is sufficient to explain the observed maintenance of chromosome copy during asexual reproduction.

Selective Constraint on Copy Number Variation in Human Piwi-Interacting RNA Loci

Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNA found in animals. PiRNAs are primarily expressed in the germline where their best understood function is to repress transposable elements. Unlike previous studies that investigated the evolution of piRNA-generating loci at the level of nucleotide substitutions, here we studied the evolution of piRNA-generating loci at the level of copy number variation (i.e. duplications and deletions) using genome-wide copy number variation data from three human populations. Our analysis shows that at the level of copy number variation there is strong selective constraint and a very high mutation rate in human piRNA-generating loci. Our results differ from a model of positive selection on copy number variation in piRNA-generating loci previously proposed in rodents. We discuss possible reasons for this difference based on the transposable element insertion histories in the rodent and primate lineages.

Abstract 2159: Genomic characterization of benign and malignant thyroid tumors

Abstract Background: Thyroid cancer cells typically harbor multiple genomic aberrations that drive malignant transformation. The goal of this study was to identify novel genetic markers to improve preoperative discrimination between benign and malignant thyroid neoplasms. Methods: Using Illumina HumanHap550 BeadChip with 561,446 single-nucleotide polymorphism (SNP) mark¬ers, we estimated copy number (CN) across the genome on 39 thyroid tumors with matched normal tissue, which included 14 benign tumors (follicular adenomas, FAs) and 25 malignant tumors (13 follicular variant papillary thyroid cancers, FVPTCs, and 12 papillary thyroid cancers, PTCs). The Cir¬cu¬lar Binary Seg¬men¬ta¬tion (CBS) algo¬rithm was applied to calculate copy number variation (CNV) based on Log R Ratio (LRR) values. Loss of heterozygosity (LOH) analysis was performed by using LOH Score plug-in in BeadStudio program. Within the regions showing CNV, candidate genes were further examined by real-time PCR. Results: The comprehensive genetic profile of 39 thyroid tumors was defined, and tumor -specific somatic change assessed using matched normal thyroid tissue as reference. With strict control of the false discovery rate, significant alterations averaged 32 CNVs in benign FAs, and 8 in malignant thyroid cancer (PTCs and FVPTCs), including 17 gains in FAs and 3 gains in cancers, and 2.9 losses in FAs and 4.4 losses in cancers. Additionally, an average of 12 copy-neutral LOHs per FA and 1 LOH per cancer were detected. Among the CNVs, we found 60 focal alterations that were specific either for benign FAs, or for thyroid cancers. We used empirical Bayes modified ANOVA to identify genes for which copy number was associated with tumor type. Increases in copy number on chromosome 12 were much more common in FAs than in cancers. GDF3, a candidate gene located in 12p13.31, was further validated by quantitative real time PCR, which showed results consistent with our SNP array results. Overall, follicular adenomas showed considerably higher levels of copy number variation than the two malignant tumor subtypes studied, i.e. PTCs and FVPTCs. Conclusions: Benign and malignant thyroid tumors may possess characteristic and distinguishable genomic profiles. The genetic changes identified in this study may allow the development of biomarkers for differentiation of benign and malignant thyroid nodules. In the next phase of this study, we will perform a transcriptome-wide expression analysis on the same tissue sample set used for the SNP array study, and perform an integrated analysis combining DNA genetic alterations and differential gene expression to further refine the identification of candidate molecular changes that may be used in differentiating benign from malignant thyroid lesions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2159.

Maize Inbreds Exhibit High Levels of Copy Number Variation (CNV) and Presence/Absence Variation (PAV) in Genome Content

Following the domestication of maize over the past ∼10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop.

Genomic structural variation and schizophrenia

It has recently been demonstrated that a large amount of structural variation exists in the human genome. Since 2004, when two landmark studies reported polymorphic levels of copy number variation in phenotypically normal individuals, our understanding of genome-wide levels of copy number variation has grown. This has inspired hypotheses about this class of variation's contribution to complex genetic phenotypes, including the specific hypothesis that structural variation is associated with psychiatric illness. The technology to accurately and efficiently detect polymorphic structural variants is still largely under development, but some examples of genomic imbalance contributing to schizophrenia and bipolar disorder already have been identified. Although much optimism surrounds this burgeoning field, the technical challenges in reliably identifying structural variation mean recent literature should be approached with caution.