Gene Copy Number Variation Research Articles

Additional value of red blood cell parameters in predicting uncommon α-thalassemia; experience from 10 years of α-globin gene sequencing and copy number variation analysis.

The diagnosis of rare forms of α-thalassemia requires laborious genetic analyses. Accurate sample selection for such evaluation is therefore essential. The main objectives of this study were to investigate the predictive power of red blood cell parameters to detect rare forms of α-thalassemia (substudy 1), and to explore the frequency of rare versus common forms of α-thalassemia in our sample population (substudy 2). In substudy 1, we reviewed all blood samples selected for extended α-hemoglobinopathy evaluation at our laboratory during 2011-2020 (n= 1217), which included DNA sequencing and/or copy number variation analysis. We assessed α-thalassemia positive samples at different levels of mean corpuscular hemoglobin (MCH) alone and in combination with results for red blood cell count (RBC) or red cell distribution width (RDW). In substudy 2, we examined the distribution of α-thalassemia genotypes for all samples submitted to a first-tier hemoglobinopathy evaluation at our laboratory during 2014-2020 (n= 6495). In substudy 1, both RBC and RDW added predictive value in detecting rare forms of α-thalassemia in samples from adults and children. In adult samples with MCH ≤ 23 pg, the presence of erythrocytosis increased the detection rate from 27% to 74% as compared to non-erythrocytosis, while normal RDW increased the detection rate from 36% to 86% as compared to elevated RDW. In substudy 2, rare forms of α-thalassemia were detected in 12% of α-thalassemia positive samples. Initial assessment of MCH, RBC, and RDW provided valuable predictive information about the presence of rare forms of α-thalassemia during hemoglobinopathy evaluation.

A Comprehensive Analysis of the Effects of Key Mitophagy Genes on the Progression and Prognosis of Lung Adenocarcinoma.

The aim of our study was to perform a comprehensive analysis of the gene expression, copy number variation (CNV) and mutation of key mitophagy genes in the progression and prognosis of lung adenocarcinoma (LUAD). We obtained the data from The Cancer Genome Atlas (TCGA). Clustering analysis was performed to stratify the mitophagy related groups. The least absolute shrinkage and selection operator (LASSO) based cox model was used to select hub survival genes. An independent validation cohort was retrieved from Gene Expression Omnibus database. We found 24 out of 27 mitophagy genes were aberrantly expressed between tumor and normal samples. CNV gains were associated with higher expression of mitophagy genes in 23 of 27 mitophagy genes. The clustering analysis identified high and low risk mitophagy groups with distinct survival differences. The high risk mitophagy groups had higher tumor mutation burden, stemness phenotype, total CNVs and lower CD4+ T cells infiltration. Drugs targeted to high risk mitophagy groups were identified including the PI3K/AKT/mTOR inhibitor, HDAC inhibitor and chemotherapy agents such as cisplatin and gemcitabine. In addition, the differentially expressed genes (DEGs) were identified between mitophagy groups. Further univariate Cox analysis of each DEG and subsequent LASSO-based Cox model revealed a mitophagy-related prognostic signature. The risk score model of this signature showed a strong ability to predict the overall survival of LUAD patients in training and validation datasets. In conclusion, the mitophagy genes played an important role in the progression and prognosis of LUAD, which might provide useful information for the treatment of LUAD.

Association between Carbonic Anhydrase VI Gene Copy Number Variations and Dental Caries Experience

The current study examined the association between the carbonic anhydrase VI (CA VI) copy number variations (CNVs) and dental caries experience in adults. In total, 202 of 35–72 years old subjects participating in the Lithuanian National Oral Health Survey (LNOHS) agreed to provide saliva samples; thus, their data were included in the current study. Information about sociodemographic, environmental, and behavioural determinants was acquired via the self-administered World Health Organization (WHO) questionnaire. Fluoride levels in the drinking water were recorded based on information provided by water suppliers. Dental caries experience was recorded by one calibrated examiner using the WHO criteria for recording caries on smooth (including proximal, buccal, and oral) or occlusal surfaces. Caries experience was measured as the total number of decayed (D₃), missing (M), filled (F) surfaces. DNA was extracted from saliva samples to examine CA VI CNVs using the QX200 Droplet Digital PCR system. Negative binomial regression and Poisson regression analyses were employed for data analyses. Based on multivariable regression analyses, higher copy number of CA VI were associated with higher caries experience on smooth surfaces (IRR 1.04, 95% CI: 1.005–1.08) and occlusal surfaces (IRR 1.02, 95% CI: 1.003–1.04). Positive associations between higher copy number of CA VI and higher caries experience on smooth and occlusal surfaces were found, suggesting that the CA VI coding gene may be associated with caries development. Future studies are needed to validate our results and to examine the underlying mechanisms of such associations.

Ten-year persistence and evolution of Plasmodium falciparum antifolate and anti-sulfonamide resistance markers pfdhfr and pfdhps in three Asian countries.

The amplification of GTP cyclohydrolase 1 (pfgch1) in Plasmodium falciparum has been linked to the upregulation of the pfdhfr and pfdhps genes associated with resistance to the antimalarial drug sulfadoxine-pyrimethamine. During the 1990s and 2000s, sulfadoxine-pyrimethamine was withdrawn from use as first-line treatment in southeast Asia due to clinical drug resistance. This study assessed the temporal and geographic changes in the prevalence of pfdhfr and pfdhps gene mutations and pfgch1 amplification a decade after sulfadoxine-pyrimethamine had no longer been widely used. A total of 536 P. falciparum isolates collected from clinical trials in Thailand, Cambodia, and Lao PDR between 2008 and 2018 were assayed. Single nucleotide polymorphisms of the pfdhfr and pfdhps genes were analyzed using nested PCR and Sanger sequencing. Gene copy number variations of pfgch1 were investigated using real-time polymerase chain reaction assay. Sequences of the pfdhfr and pfdhps genes were obtained from 96% (517/536) and 91% (486/536) of the samples, respectively. There were 59 distinct haplotypes, including single to octuple mutations. The two major haplotypes observed included IRNI-AGEAA (25%) and IRNL-SGKGA (19%). The sextuple mutation IRNL-SGKGA increased markedly over time in several study sites, including Pailin, Preah Vihear, Ratanakiri, and Ubon Ratchathani, whereas IRNI-AGEAA decreased over time in Preah Vihear, Champasak, and Ubon Ratchathani. Octuple mutations were first observed in west Cambodia in 2011 and subsequently in northeast Cambodia, as well as in southern Laos by 2018. Amplification of the pfgch1 gene increased over time across the region, particularly in northeast Thailand close to the border with Laos and Cambodia. Despite the fact that SP therapy was discontinued in Thailand, Cambodia, and Laos decades ago, parasites retained the pfdhfr and pfdhps mutations. Numerous haplotypes were found to be prevalent among the parasites. Frequent monitoring of pfdhfr and pfdhps in these areas is required due to the relatively rapid evolution of mutation patterns.

156. Using Whole Genome Sequencing to Genetically Profile and Analyze Escherichia coli Isolates with Varying Resistance to β-Lactam/β-lactamase Inhibitor Combinations

Abstract Background Whole-genome sequencing has gained interest for assessing antimicrobial resistance (AMR). However, multiple studies have shown a poor correlation between the β-lactam/β-lactamase inhibitor (BL/BLI) phenotype and β-lactamase gene presence/absence. In a recent publication, we found that amplification of β-lactamase encoding genes potentially contribute to BL/BLI resistance and here we explore if gene amplifications allow for better BL/BLI genotype/phenotype correlations. Methods We selected 109 E. coli bacteremia isolates and performed ETests (bioMerieux, Inc) to determine AST profiles using the following BL/BLI antibiotics: ampicillin/sulbactam (SAM), amoxicillin/clavulanic acid (AMC), and piperacillin/tazobactam (TZP). CLSI M100 guidelines (2018) were used to stratify isolates into 4 groups: SAM/AMC/TZP susceptible (Group 1), SAM resistant only (Group 2), SAM/AMC resistant (Group 3), and SAM/AMC/TZP resistant (Group 4). Short-read whole genome sequencing was performed on these isolates and analyzed by identifying AMR genes, establishing multi-locus sequence type (MLST), estimating copy number variants (CNV), and investigating blaTEM-1 promoter regions in relation to phenotype. A t-Distributed Stochastic Neighbor Embedding (t-SNE) clustering method and a core-genome, maximum-likelihood phylogeny was generated to group isolates based on AMR gene content and CNV data. Results We found 34 different MLST groups with the three most common sequence types ST131 (n=41), ST1193 (n=12), and ST648 (n=6). Group 1 and Group 2 isolates had similar copy number estimates of β-lactamase blaTEM-1 (1.8X and 1.5X respectively) while there was an increase for both Group 3 (3.7X) and Group 4 (8.2X) isolates (Table 1). Additionally, blaOXA-1 was primarily present in Group 4 isolates (63%) with elevated copy numbers (5.2X). We did not observe associations of blaTEM-1 promoter regions with each of the BL/BLI Groups. Our t-SNE analysis shows that Group 1, Group 3, and Group 4 isolates cluster independently. Core Genome Maximum-likelihood Phylogeny. Midpoint-rooted core-genome maximum-likelihood phylogeny comparing core elements (MLST) to non core elements (CNV). AMR CNV and presence/absence associate better with BL/BLI phenotypes than do core elements. Conclusion There is a clear delineation between fully susceptible and fully resistant BL/BLI E. coli isolates when gene amplification and presence/absence of narrow-spectrum β-lactamases are considered, which should be considered for BL/BLI resistance prediction models. Disclosures David E. Greenberg, MD, Shionogi: Grant/Research Support.

Chloroplast genome assemblies and comparative analyses of commercially important Vaccinium berry crops

Vaccinium is a large genus of shrubs that includes a handful of economically important berry crops. Given the numerous hybridizations and polyploidization events, the taxonomy of this genus has remained the subject of long debate. In addition, berries and berry-based products are liable to adulteration, either fraudulent or unintentional due to misidentification of species. The availability of more genomic information could help achieve higher phylogenetic resolution for the genus, provide molecular markers for berry crops identification, and a framework for efficient genetic engineering of chloroplasts. Therefore, in this study we assembled five Vaccinium chloroplast sequences representing the economically relevant berry types: northern highbush blueberry (V. corymbosum), southern highbush blueberry (V. corymbosum hybrids), rabbiteye blueberry (V. virgatum), lowbush blueberry (V. angustifolium), and bilberry (V. myrtillus). Comparative analyses showed that the Vaccinium chloroplast genomes exhibited an overall highly conserved synteny and sequence identity among them. Polymorphic regions included the expansion/contraction of inverted repeats, gene copy number variation, simple sequence repeats, indels, and single nucleotide polymorphisms. Based on their in silico discrimination power, we suggested variants that could be developed into molecular markers for berry crops identification. Phylogenetic analysis revealed multiple origins of highbush blueberry plastomes, likely due to the hybridization events that occurred during northern and southern highbush blueberry domestication.

Exploring biomarkers for prognosis and neoadjuvant chemosensitivity in rectal cancer: Multi-omics and ctDNA sequencing collaboration.

This study aimed to identified the key genes and sequencing metrics for predicting prognosis and efficacy of neoadjuvant chemotherapy (nCT) in rectal cancer (RC) based on genomic DNA sequencing in samples with different origin and multi-omics association database. We collected 16 RC patients and obtained DNA sequencing data from cancer tissues and plasma cell-free DNA before and after nCT. Various gene variations were analyzed, including single nucleotide variants (SNV), copy number variation (CNV), tumor mutation burden (TMB), copy number instability (CNI) and mutant-allele tumor heterogeneity (MATH). We also identified genes by which CNV level can differentiate the response to nCT. The Cancer Genome Atlas database and the Clinical Proteomic Tumor Analysis Consortium database were used to further evaluate the specific role of therapeutic relevant genes and screen out the key genes in multi-omics levels. After the intersection of the screened genes from differential expression analysis, survival analysis and principal components analysis dimensionality reduction cluster analysis, the key genes were finally identified. The genes CNV level of principal component genes in baseline blood and cancer tissues could significantly distinguish the two groups of patients. The CNV of HSP90AA1, EGFR, SRC, MTOR, etc. were relatively gained in the better group compared with the poor group in baseline blood. The CNI and TMB was significantly different between the two groups. The increased expression of HSP90AA1, EGFR, and SRC was associated with increased sensitivity to multiple chemotherapeutic drugs. The nCT predictive score obtained by therapeutic relevant genes could be a potential prognostic indicator, and the combination with TMB could further refine prognostic prediction for patients. After a series of analysis in multi-omics association database, EGFR and HSP90AA1 with significant differences in multiple aspects were identified as the key predictive genes related to prognosis and the sensitivity of nCT. This work revealed that effective combined application and analysis in multi-omics data are critical to search for predictive biomarkers. The key genes EGFR and HSP90AA1 could serve as an effective biomarker to predict prognose and neoadjuvant chemosensitivity.

Detection of the Copy Number Variants of Genes in Patients with Familial Cardiac Diseases by Massively Parallel Sequencing.

The implication of copy number variations in familial heart disease is known, although in-depth knowledge is lacking; hence, more studies are needed to further our understanding. Massively parallel sequencing, thanks to its recent surge in use, is emerging as a valid tool for the detection of this type of variant, through the use of appropriate software. We conducted a study with 182 patients diagnosed with mendelian cardiovascular diseases who underwent sequencing using a cardiac gene panel and then a specific calling process for copy number variations (CNVs) with ExomeDepth software, which provides us with a Bayes factor (BF), a score of the probability that a CNV detected is true. After a rigorous CNV prioritization process, we confirmed the variants obtained by MLPA or SNP-based array, finding three real CNVs in five individuals in the MYH11, FBN1 and PDMI7 genes. The confirmed CNVs present in all cases BF values >60, thus establishing a threshold to consider real CNVs in the calling process carried out by ExomeDepth on our gene panel.

Genotyping Microbial Communities with MIDAS2: From Metagenomic Reads to Allele Tables.

The Metagenomic Intra-Species Diversity Analysis System 2 (MIDAS2) is a scalable pipeline that identifies single nucleotide variants and gene copy number variants in metagenomes using comprehensive reference databases built from public microbial genome collections (metagenotyping). MIDAS2 is the first metagenotyping tool with functionality to control metagenomic read mapping filters and to customize the reference database to the microbial community, features that improve the precision and recall of detected variants. In this article we present four basic protocols for the most common use cases of MIDAS2, along with supporting protocols for installation and use. In addition, we provide in-depth guidance on adjusting command line parameters, editing the reference database, optimizing hardware utilization, and understanding the metagenotyping results. All the steps of metagenotyping, from raw sequencing reads to population genetic analysis, are demonstrated with example data in two downloadable sequencing libraries of single-end metagenomic reads representing a mixture of multiple bacterial species. This set of protocols empowers users to accurately genotype hundreds of species in thousands of samples, providing rich genetic data for studying the evolution and strain-level ecology of microbial communities. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Species prescreening Basic Protocol 2: Download MIDAS reference database Basic Protocol 3: Population single nucleotide variant calling Basic Protocol 4: Pan-genome copy number variant calling Support Protocol 1: Installing MIDAS2 Support Protocol 2: Command line inputs Support Protocol 3: Metagenotyping with a custom collection of genomes Support Protocol 4: Metagenotyping with advanced parameters.

Association of Fc Gamma Receptor 3B Gene Copy Number Variation with Rheumatoid Arthritis Susceptibility.

Structural variations such as copy number variants (CNVs) have been associated with multiple autoimmune diseases. In this study, we explored the association of the Fc gamma receptor 3B gene (FCGR3B) copy number variation (CNV) with rheumatoid arthritis (RA) susceptibility and related serological traits in the Pakistani population. We also performed a meta-analysis of four published FCGR3B CNV studies along with the current study. A total of 927 subjects (597 RA cases, 330 healthy controls) were recruited from three rheumatology centers in Pakistan. Anti-cyclic citrullinated peptide (anti-CCP) antibodies and rheumatoid factor (RF) were measured in RA patients. FCGR3B copy number was assayed using the TaqMan® CN assay (Hs04211858_cn, Applied Biosystems, Foster City, CA, USA) and the copy number was estimated by using CopyCaller® software (version 2.1; Applied Biosystems, USA). Logistic regression was applied to calculate the odds ratio (OR) of RA risk associated with FCGR3B CNV using sex and age as covariates in R. Meta-analysis on four previously published studies and the current study was performed using the random-effect model. We observed a significant association between FCGR3B copy number < 2 and RA susceptibility (OR = 1.53; 95% CI: 1.05 to 2.22; p = 0.0259) and anti-CCP seropositivity (OR 2.56; 95% CI: 1.34 to 4.89; p = 0.0045). A non-significant association of FCGR3B copy number < 2 was also observed between increased rheumatoid factor (RF) seropositivity (OR = 1.74; 95% CI:0.93 to 3.26; p = 0.0816). Meta-analysis on 13,915 subjects (7005 RA cases and 6907 controls) also showed significant association of copy number < 2 with the increased risk of RA (OR = 1.30; 95% CI: 1.07 to 1.56; p = 0.00671). FCGR3B copy number < 2 is associated with increased RA risk and anti-CCP seropositivity.

Genetic Alterations of Melanoma Brain Metastases: A Systematic Review and Meta-Analysis.

Data on molecular alterations harbored by melanoma brain metastases (MBMs) are limited, and this has hampered the development of more effective therapeutic strategies. We conducted a systematic review and meta-analysis of all the studies reporting DNA sequencing data of MBMs, in order to identify recurrently mutated genes and molecular pathways significantly enriched for genetic alterations. We searched PubMed, Embase and Scopus for articles published from the inception of each database to June 30, 2021. We included in the analysis all the studies that reported individual patient data on DNA sequencing of MBMs, assessing single nucleotide variants (SNVs) and/or gene copy number variations (CNVs) in at least five tumor samples. Meta-analysis was performed for genes evaluated for SNVs and/or CNVs in at least two studies. Pooled proportions of samples with SNVs and/or CNVs was calculated by applying random-effect models based on the DerSimonian-Laird method. Gene-set enrichment analysis (GSEA) was performed to identify molecular pathways significantly enriched for mutated genes. Ten studies fulfilled the inclusion criteria and were included in the analysis, for a total of 531 samples of MBMs evaluated. Twenty-seven genes were found recurrently mutated with a meta-analytic rate of SNVs higher than 5%. GSEA conducted on the list of these 27 recurrently mutated genes revealed vascular endothelial growth factor-activated receptor activity and transmembrane receptor protein tyrosine kinase activity to be among the top 10 gene ontology (GO) molecular functions significantly enriched for mutated genes, while regulation of apoptosis and cell proliferation were among the top 10 significantly enriched GO biological processes. Notably, a high meta-analytic rate of SNVs was found in several actionable cancer-associated genes, such as all the vascular endothelial growth factor (VEGF) receptor isoforms (i.e., Flt1 and Flt2 genes, for both SNV rate: 0.22, 95% CI 0.04-0.49; KDR gene, SNV rate: 0.1, 95% CI 0.05-0.16). Finally, two tumor suppressor genes were characterized by a high meta-analytic rate of CNVs: CDKN2A/B (CNV rate: 0.59, 95% CI 0.23-0.90) and PTEN (CNV rate: 0.31, 95% CI 0.02-0.95). MBMs harbored actionable molecular alterations that could be exploited as therapeutic targets to improve the poor prognosis of patients.

Frequent spontaneous structural rearrangements promote rapid genome diversification in a Brassica napus F1 generation.

In a cross between two homozygous Brassica napus plants of synthetic and natural origin, we demonstrate that novel structural genome variants from the synthetic parent cause immediate genome diversification among F1 offspring. Long read sequencing in twelve F1 sister plants revealed five large-scale structural rearrangements where both parents carried different homozygous alleles but the heterozygous F1 genomes were not identical heterozygotes as expected. Such spontaneous rearrangements were part of homoeologous exchanges or segmental deletions and were identified in different, individual F1 plants. The variants caused deletions, gene copy-number variations, diverging methylation patterns and other structural changes in large numbers of genes and may have been causal for unexpected phenotypic variation between individual F1 sister plants, for example strong divergence of plant height and leaf area. This example supports the hypothesis that spontaneous de novo structural rearrangements after de novo polyploidization can rapidly overcome intense allopolyploidization bottlenecks to re-expand crops genetic diversity for ecogeographical expansion and human selection. The findings imply that natural genome restructuring in allopolyploid plants from interspecific hybridization, a common approach in plant breeding, can have a considerably more drastic impact on genetic diversity in agricultural ecosystems than extremely precise, biotechnological genome modifications.

Association between Fc-Gamma Receptor Polymorphisms and Autoimmune Neutropenia in Early Childhood in Danish Patients

Background Copy number variation (CNV) in the genome is difficult to analyze with standard genotyping methods. CNV in the Fcγ receptor region (FCGR) is associated with risk of autoimmune disease. Genetic variation in Fc-gamma coding genes is also abundant, and several functional polymorphisms are described. Autoimmune neutropenia (AIN) primarily affects children under the age of five but can also affect adults. The disease is caused by autoantibodies reactive to neutrophil surface antigens, primarily the FCGR3B receptor. We investigated the association between common SNPs and CNV in Fcγ receptors and AIN. Aims Establish the association between CNV and genetic variation in Fc-gamma receptor coding genes in Danish patients. Methods 127 Danish AIN cases were compared with 610 healthy and unrelated Danish blood donors for FCGR2A-H166R, and with 919 Caucasians (1) for FCGR3A-V158F, FCGR2B-B2.4, FCGR2B-I232T and CNV. Molecular determination of copy number and genotypes was determined using Multiplex Ligation Probe Assay (MLPA) with the following SALSA MLPA probemixes: P110-C1 FCGR mix 1 and P111-C1 FCGR mix 2. Statistical analysis was performed with Fisher Exact test. Results The polymorphisms FCGR2A-166H, FCGR3A-158F, FCGR2B-232I were statistically significantly associated with a higher risk of AIN (Table 1). The presence of a FCGR2B-2B.4 promoter haplotype was associated with a significantly lower risk of AIN (OR=0.5). There was not observed a difference in the distribution of CNV for the Fc-gamma receptor genes (data not shown). Summary/Conclusion In our Danish cohort, we report an association between AIN and polymorphisms in Fc-gamma receptor 2A, 3A and 2B, indicating a significance of other Fc-gamma receptors than FCGR3B. We do not observe a difference in the distribution of CNV of the Fc-gamma receptor genes. 1) Nagelkerke SQ, Tacke CE, Breunis WB, Tanck MWT, Geissler J, Png E, Hoang LT, van der Heijden J, Naim ANM, Yeung RSM, Levin ML, Wright VJ, Burgner DP, Ponsonby A-L, Ellis JA, Cimaz R, Shimizu C, Burns JC, Fijnvandraat K, van der Schoot CE, van den Berg TK, de Boer M, Davila S, Hibberd ML,Kuijpers TW and the International Kawasaki Disease Genetics Consortium (2019) Extensive Ethnic Variation and Linkage Disequilibrium at the CGR2/3 Locus: Different Genetic Associations Revealed in Kawasaki Disease. Front. Immunol. 10:185. doi: 10.3389/fimmu.2019.00185 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Geometric Network Analysis Defines Poor-Prognosis Subtypes in Multiple Myeloma

The prognosis for patients with multiple myeloma (MM) is dependent on a variety of measures including treatment received, clinical factors, and genomic characteristics, with several specific genomic features predicting for shorter progression free survival (PFS). Studies have been conducted to identify biomarkers using gene expression profiles and copy number variation (CNV) data. However, no study to date has integrated genomic data from a systems view, incorporating interactions between biomarkers in a network. We apply an innovative geometric network analysis that integrates complex interactions to characterize patterns of biological behavior not captured by individual genomic events. The methodology is mathematically well-defined and has no fitting parameters. We hypothesized that such a systems mathematical approach applied to gene interaction networks may delineate biologically relevant MM subtypes and potential new therapeutic targets. We overlaid RNA-Seq and CNV data from the MMRF CoMMpass dataset (IA19) on a gene interactome derived from the Human Protein Reference Database (HPRD) using a novel graph metric of network robustness - Ollivier-Ricci curvature (ORC). ORC considers both local and global connectivity in assessing the robustness of each pathway characterized by feedback loops in a network. Hierarchical agglomerative clustering of the resulting ORC matrix computed for each omics-dataset was performed to identify subtypes based on the patterns of curvature, with the optimal number of clusters determined by silhouette score. Survival analysis for progression free survival (PFS) was performed employing Kaplan-Meier and log-rank tests. A differential gene expression analysis between high and low risk groups, as identified by the gene expression data, was then carried out using raw read counts with DESeq2. Further downstream bioinformatic analysis was conducted using MetaCore software. Both RNA-Seq and CNV data were available in 659 patients. Gene product interactions were determined by protein-protein interactions derived from HPRD, resulting in a network with 8,468 nodes and 33,695 edges. The ORC analysis using RNA-Seq data resulted in 6 clusters, and Kaplan-Meier analysis showed statistical significance in PFS with log-rank p=0.0016. Clusters predicting the best and worst PFS were associated with specific genomic features including CCND1 and MAF/MAFB translocations, respectively (each p<0.05, Fisher's exact test). Considering the central role of CCND1 in MM biology, we repeated our analysis with t(11;14) patients excluded, with the resulting clustering remaining significant for predicting PFS. The ORC analysis of CNV data on HPRD resulted in 8 clusters, and Kaplan-Meier analysis showed statistical significance in PFS with log-rank p=0.0082. Genomic features associated with the clusters included hyperdiploidy and CCND1 (longest PFS), and MAF-translocations and APOBEC-activity (shortest PFS) (each p<0.05, Fisher's). The worst PFS cluster had a higher prevalence of chromothripsis (p<0.0001, Fisher's) than other clusters, known to be an independent predictor for MM survival. Considering the impact of hyperdiploidy on CNV in MM, analyzing hyperploid and non-hyperdiploid patients separately was still significant for PFS prediction. After DESeq2 analysis to perform differential gene expression analysis between the high and low risk groups, the Benjamini-Hochberg method was applied to adjust the resulting p-values, producing false discovery rate (FDR). A list of 118 genes derived with FDR < 0.05 and |fold change| > 3.5 was input into MetaCore to identify biological correlates associated with the risk of PFS. Key implicated biological processes included cell cycle progression, inflammation and immune response. In this study, we applied the geometric network analysis tool ORC to multi-omics data in MM represented as biological networks to identify individuals at high risk of short PFS and relevant biological correlates. One hundred and eighteen key genes associated with MM prognosis were identified. Decreased robustness of signaling near immune-related genes was associated with shorter survival. For both RNAseq and CNV-based data, patient clusters were associated with known predictors of PFS, and remained predictive when removing specific genomic features, indicating that our methods are providing new biological insights. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Value of Gene Copy Number Variation Detection in Peripheral Blood in the Diagnosis and Treatment of Lymphoma

Objective To investigate the consistency between the results of gene copy number variation (CNA) in peripheral blood and bone marrow or tissue samples of lymphoma patients with curative and its clinical value in diagnosis and treatment. Methods A total of 5 patients with lymphoma were selected. Case 1 was diffuse large B-cell lymphoma (DLBCL) without bone marrow invasion. Tissue samples and peripheral blood samples were collected at the same time point before treatment. Case 2 was high-grade B-cell lymphoma (HGBL) stage IV, tissue samples were collected before treatment at the initial diagnosis and peripheral blood samples were collected after treatment. Case 3 was DLBCLIV, tissue samples were collected at initial diagnosis and peripheral blood samples were collected during remission after treatment. Case 4 was DLBCL-IV B stage. Peripheral blood samples were collected before treatment at initial diagnosis and bone marrow samples were collected during remission after treatment. Case 5 was a case of hepatosplenic T-cell lymphoma (HSTL). Tissue samples before treatment and peripheral blood samples after treatment were collected at initial diagnosis and analyzed in combination with clinical efficacy. Results 2p -, 2q-, 3p-, 4p -, 6q-, 9p+, 10q-, 11q+, 19p + and 19q+ chromosome abnormalities were detected in the tissues and peripheral blood CNA of case 1, and the 3p -, 6q-, 9p +, 11q+ and 19q+ abnormalities were consistent. In case 2, 4p -, 9P +, 9p-, 16p+, 16q- and +X gene variants were detected in peripheral blood CNA than in tissue CNA at initial diagnosis. In case 3, obvious abnormal signals were detected in tissues at initial diagnosis, but no abnormal signals were detected in peripheral blood samples during remission. In case 4, obvious abnormal signals were detected in peripheral blood at initial diagnosis, but no abnormal signals were detected in bone marrow during remission. In case 5, obvious abnormal signals were detected in tissues at initial diagnosis, and a small amount of abnormal signals were detected in peripheral blood after treatment. The results of CNA detection in 5 patients were consistent with the clinical efficacy. All 3 DLBCL patients showed 6q-chromosome abnormalities in CNA test before treatment. Conclusion The results of CNA detection in peripheral blood are consistent with those in tissue and bone marrow. The dynamic monitoring results of in peripheral blood CNA can be used as a basis for monitoring the curative effect of lymphoma.

A novel cuproptosis-related gene signature of prognosis and immune microenvironment in head and neck squamous cell carcinoma cancer.

Cuproptosis is a novel form of cell death that is highly related to mitochondrial metabolism and mediated by protein lipoacetylation. This study systematically assessed the differential expression and genetic alterations of cuproptosis-related genes (CRGs) in head and neck squamous-cell carcinoma (HNSCC) and constructed CRG risk models to predict survival in patients with HNSCC. We investigated the expression of 19 CRGs in HNSCC and noncancerous tissues, and the relationship between mutation load, immune infiltration, and clinical features was examined based on data from public databases. CRG risk models were constructed by univariate Cox analysis and lasso regression and validated by independent datasets for their accuracy in predicting survival outcomes in patients with HNSCC. The expression distribution of CRGs in HNSCC cells was further explored in the HNSCC single-cell sequencing dataset. NFE2L2, ATP7A, FDX1, LIAS, DLD, DLAT, PDHB, MTF1 and DBT were highly expressed in noncancerous samples, while GLS, CDKN2A and DLST were highly expressed in HNSCC samples (p < 0.05). Gene copy number variation frequency (CNV) revealed CDKN2A, FDX1 and DLAT copy number deletions and LIPT2 and NFE2L2 copy number increases. Ten CRGs were used to construct a risk model to predict overall survival (OS) in HNSCC, yielding reduced OS in the high-risk group compared to the low-risk group, training group (p = 9.733e - 05), and testing group (p = 0.040). The CRG risk model was significantly correlated with various immune cells, regulatory T cells (Tregs) and memory B cells were significantly negatively correlated (p = 0.027, p = 0.00084), and resting CD4 memory T cells was significantly positively correlated (p = 9e - 04). Most CRGs significantly affected the clinical characteristics of HNSCC. NFE2L2, SLC31A1, PDHA1, CDKN2A and DBT were highly expressed in epithelial cells of HNSCC, while SLC31A1, DBT and NFE2L2 were highly expressed in T cells, and SLC31A1 in B cells. In monocytes, NFE2L2, SLC31A1 and PDHA1 were highly expressed. The CRG risk model can be used as a potential prognostic factor for HNSCC patients and may provide new insights into cancer treatment from the perspective of copper metabolism.

Integrated bioinformatics analysis and experimental validation reveals fatty acid metabolism-related prognostic signature and immune responses for uterine corpus endometrial carcinoma.

Uterine corpus endometrial carcinoma (UCEC) is the third most common gynecologic malignancy. Fatty acid metabolism (FAM) is an essential metabolic process in the immune microenvironment that occurs reprogramming in the presence of tumor signaling and nutrient competition. This study aimed to identify the fatty acid metabolism-related genes (FAMGs) to develop a risk signature for predicting UCEC. The differentially expressed FAMGs between UCEC samples and controls from TCGA database were discovered. A prognostic signature was then constructed by univariate, least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analyses. Based on the median risk score, UCEC samples were categorized into high- and low-FAMGs groups. Kaplan-Meier (K-M) curve was applied to determine patients' overall survival (OS). The independent prognostic value was assessed by uni- and multivariate analyses. The associations between the risk score and immune status, immune score, and drug resistance were evaluated. Quantitative Real-time PCR (qRT-PCR) was utilized to confirm FAMGs expression levels in UCEC cells. We built a 10-FAMGs prognostic signature and examined the gene mutation and copy number variations (CNV). Patients with a high-FAMGs had a worse prognosis compared to low-FAMGs patients in TCGA train and test sets. We demonstrated that FAMGs-based risk signature was a significant independent prognostic predictor of UCEC. A nomogram was also created incorporating this risk model and clinicopathological features, with high prognostic performance for UCEC. The immune status of each group was varied, and immune score was higher in a low-FAMGs group. HLA-related genes such as DRB1, DMA, DMB, and DQB2 had higher expression levels in the low-FAMGs group. Meanwhile, high-FAMGs patients were likely to response more strongly to the targeted drugs Bortezomib, Foretinib and Gefitinib. The qRT-PCR evidence further verified the significant expression of FAMGs in this signature. A FAMGs-based risk signature might be considered as an independent prognostic indicator to predict UCEC prognosis, evaluate immune status and provide a new direction for therapeutic strategies.

Abstract 15501: Rare Genomic Copy Number Variants Implicate New Candidate Genes for Bicuspid Aortic Valve

Introduction: Bicuspid Aortic Valve (BAV), the most common adult congenital heart defect, is a major cause of aortic insufficiency or stenosis requiring valve replacement and thoracic aortic aneurysms predisposing to acute aortic dissections. The spectrum of BAV ranges from early onset valve and aortic complications to sporadic late onset disease. We determined the frequency and gene content of rare genomic copy number variants (CNVs) in isolated BAV cases. Methods: We performed genome-wide SNP microarray analysis of familial BAV cases with early onset complications in the UTHealth BAV Research Registry (EBAV, n=394) and elderly probands from the International BAV Consortium (BAVCON, n=4216). CNVs were detected in Illumina genotypes using the PennCNV, QuantiSNP and cnvPartition algorithms. BAV cases were compared to 16,576 controls without known cardiovascular disease from the Database of Genotypes and Phenotypes. Only CNV calls with &gt;6 consecutive variants, &gt;20 kb in length, identified by at least two algorithms and intersected with known genes were included. For comparison, we assessed a cohort with left ventricular outflow tract obstructive lesions including BAV (LVOTO, n=1561) using identical methods. CNV burden and associations were tested using PLINK. Results: We identified 84 large recurrent CNVs in BAV cases that are absent or rare (&lt;0.1%) in controls. 34 rare CNVs overlap between BAV and LVOTO cases and involve candidate genes that interact with each other during heart development. The largest and most prevalent of the recurrent CNVs are 8p23 duplications involving GATA4 (OR 340, CI: 42-2700). CNVs of genes that cause BAV when mutated were significantly enriched in cases (n=135, P =1x10 -27 ). The overall burden of rare genic CNVs, specifically large deletions, was also increased in BAV cases (empiric P &lt; 1x10 -5 ) and was higher in younger EBAV cases than in older BAVCON cases. Conclusion: We identified likely pathogenic CNVs in more than 10% of BAV cases, implicating alterations of candidate genes at these loci in the pathogenesis of BAV.

MIDAS2: Metagenomic Intra-species Diversity Analysis System.

The Metagenomic Intra-Species Diversity Analysis System (MIDAS) is a scalable metagenomic pipeline that identifies single nucleotide variants (SNVs) and gene copy number variants in microbial populations. Here, we present MIDAS2, which addresses the computational challenges presented by increasingly large reference genome databases, while adding functionality for building custom databases and leveraging paired-end reads to improve SNV accuracy. This fast and scalable reengineering of the MIDAS pipeline enables thousands of metagenomic samples to be efficiently genotyped. The source code is available at https://github.com/czbiohub/MIDAS2. Supplementary data are available at Bioinformatics online.

OR25-2 RBFOX2 Copy Number Variation Disrupts Urethral Development Causing Hypospadias

Abstract Despite the frequency of hypospadias (1: 125 live male births) often the etiology is not known. The Lamb Laboratory previously determined that RNA Binding Fox-1 Homolog 2 (RBFOX2) copy number variation (CNV) is associated with clinical cases of upper and lower tract genitourinary (GU) anomalies, including hypospadias. RBFOX2 encodes a RNA binding protein that regulates mRNA splicing. The hypothesis was tested that altered RBFOX2 gene dosage impacts the developing penis transcriptome via mRNA splicing, specifically the mesenchymal to epithelial transition critical for penile urethra formation, resulting in hypospadias.Searches, in publically available and local clinical databases, centered on hypospadias occurance with gene CNV: identified the RBFOX2 locus as a gene dosage sensitve region. To identify developmental pathways regulated by RBFOX2 in the GU system, Rbfox2 haploinsufficient and null mouse models were developed, and penises were collected for RNA-seq, in situ hybridization, and micro-CT analysis of urethral development. In situ hybridization together with re-analysis of published scRNA-seq datasets were employed to define the anatomical regions of Rbfox2-specific expression in the developing mouse penis [Amato &amp; Yao, PNAS 118(25): e2103856118, 2021; Armfield &amp; Cohn, Dev Biol 477: 145-154.2021].Publicly available clinical data identified 17 patients (0.068% n=24,896) with pathogenic RBFOX2 CNVs: 4 were noted to have hypospadias. Results from our cohort of non-syndromic GU birth defect patient samples identified 11 patients (2.4% n=450) with RBFOX2 CNVs: 21% of whom have hypospadias. RNA immunoprecipitation agianst RBFOX2 in human kidney cells validated mRNA transcipt targets previously identified in cardiac cells [Verma 2016]. Micro-CT analysis on fetal Rbfox2-haploinsufficient mice validated the modeled clinical phenotype. Combined in situhybridization and scRNA-seq spatial analysis on mouse samples revealed, after penile sex determination (E15.5), Rbfox2 expression becomes restricted to the penile urethral mesenchyme. Differential gene expression and transcript usage analysis on the Rbfox2-loss mouse penis transcriptome identified mis-expression of gene products associated the estrogen signalling pathway.Alterations in RBFOX2 gene dosage clinically correlate with upper and lower tract genitourinary anomalies, specifically hypospadias. Data from human cell lines and mouse models seemingly indicate the phenotype seems is partially the product of an RBFOX2 role in directing the mesenchymal to epithelial transition. Specifically our data support the a direct role of RBFOX2 in balancing the competing male and female pathways at the level of estrogen and androgen signaling pathways. We are currently using immunofluorescence experiments to define the changes in androgen-receptor, estrogen-receptor, and RBFOX2 patterning throughout perinatal penis development. Presentation: Monday, June 13, 2022 11:15 a.m. - 11:30 a.m.