Convergent copy number increase of genes associated with freshwater colonization in fishes.

Genome-wide Transcriptome Profiling Reveals the Functional Impact of Rare De Novo and Recurrent CNVs in Autism Spectrum Disorders

Introduction:Advancements in tumor characterization by solid and liquid biopsy are instrumental in the evolution of personalized medicine. Recent improvements in assay sensitivity for profiling DNA variants such as single nucleotide polymorphisms (SNP) and indels have facilitated expansion of both solid and liquid biopsy applications. However, measurement of copy number variations (CNV) is a relatively new application, and few reference materials exist to aid in assay development and optimization. In this study, we demonstrate for the first time the development of 17 whole gene CNV reference standards in a background of the highly characterized NIST Genome in a Bottle GM24385 genomic DNA.Method:DNA molecules containing full genes of MET, ERBB2, ERBB3, PIK3CA, EGFR, BRAF, FGFR1, FGFR2, FGFR4, KIT, KRAS, MYC-N, PDGFRA, MDM2, CD274, MYC-L and MYC were each spiked separately into background GM24385 genomic DNA to generate 2.8 copies of each gene. Copy number for each of the 17 CNV samples was determined using Bio-Rad® Droplet DigitalTM PCR (ddPCRTM). To assess linearity over a range of copy numbers, MET and ERBB2 CNV ladder reference materials with copy gains at 6 different levels 3, 6, 9, 12 and 15 copies were also generated. To mimic circulating tumor DNA, each CNV sample was fragmented and size selected to recover a population containing ~170 bp size fragments. Copy number was also verified post size selection using ddPCRTM and the Ion S5TM XL system. Multiplexed triple CNV controls with MET, EFGR and ERBB2 were also developed at 3, 6, 9, 12 and 15 copies and analyzed by ddPCRTM.Results:Copy number for all 17 genes measured at 2.80 ± 0.28 copies by ddPCR. When tested in a ctDNA copy number ladder format, MET and ERBB2 showed good linearity of R2=0.99 for observed versus expected # of copies. Good correlation between ddPCR and NGS was also observed, with a slope of 1.004 for MET and 0.969 for ERBB2 across increasing copy numbers and standard deviation (SD) of ± 10%. Notably, SD increased at copy numbers higher than 12. The average size of fragmented ctDNA CNV reference standards were observed to be ~170 bp on the Agilent 2100 Bioanalyzer system. Finally, the multiplexed triple CNV control also performed as expected on ddPCR with SD of ± 10%.Conclusion:A novel method for producing CNV and ctDNA CNV controls has been developed that enables preparation of any copy gain level with a known gDNA background. Simpler QC materials mimicking patient samples will enable simpler CNV test method development and analytical validation, which will be critical for laboratories to introduce CNV solid and liquid biopsy testing into the field.Citation Format: Kunal Banjara, Lu Zheng, Samyuktha Dasari, Kara Norman. Development of novel copy number variation (CNV) reference materials in genome in a Bottle background [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5139.

Introduction: Advancements in tumor characterization by solid and liquid biopsy are instrumental in the evolution of personalized medicine. Recent improvements in assay sensitivity for profiling DNA variants such as single nucleotide polymorphisms (SNP) and indels have facilitated expansion of both solid and liquid biopsy applications. However, measurement of copy number variations (CNV) is a relatively new application, and few reference materials exist to aid in assay development and optimization. In this study, we demonstrate for the first time the development of 17 whole gene CNV reference standards in a background of the highly characterized NIST Genome in a Bottle GM24385 genomic DNA. Method: DNA molecules containing full genes of MET, ERBB2, ERBB3, PIK3CA, EGFR, BRAF, FGFR1, FGFR2, FGFR4, KIT, KRAS, MYC-N, PDGFRA, MDM2, CD274, MYC-L and MYC were each spiked separately into background GM24385 genomic DNA to generate 2.8 copies of each gene. Copy number for each of the 17 CNV samples was determined using Bio-Rad® Droplet DigitalTM PCR (ddPCRTM). To assess linearity over a range of copy numbers, MET and ERBB2 CNV ladder reference materials with copy gains at 6 different levels 3, 6, 9, 12 and 15 copies were also generated. To mimic circulating tumor DNA, each CNV sample was fragmented and size selected to recover a population containing ~170 bp size fragments. Copy number was also verified post size selection using ddPCRTM and the Ion S5TM XL system. Multiplexed triple CNV controls with MET, EFGR and ERBB2 were also developed at 3, 6, 9, 12 and 15 copies and analyzed by ddPCRTM. Results: Copy number for all 17 genes measured at 2.80 ± 0.28 copies by ddPCR. When tested in a ctDNA copy number ladder format, MET and ERBB2 showed good linearity of R2=0.99 for observed versus expected # of copies. Good correlation between ddPCR and NGS was also observed, with a slope of 1.004 for MET and 0.969 for ERBB2 across increasing copy numbers and standard deviation (SD) of ± 10%. Notably, SD increased at copy numbers higher than 12. The average size of fragmented ctDNA CNV reference standards were observed to be ~170 bp on the Agilent 2100 Bioanalyzer system. Finally, the multiplexed triple CNV control also performed as expected on ddPCR with SD of ± 10%. Conclusion: A novel method for producing CNV and ctDNA CNV controls has been developed that enables preparation of any copy gain level with a known gDNA background. Simpler QC materials mimicking patient samples will enable simpler CNV test method development and analytical validation, which will be critical for laboratories to introduce CNV solid and liquid biopsy testing into the field. Citation Format: Kunal Banjara, Lu Zheng, Samyuktha Dasari, Kara Norman. Development of novel copy number variation (CNV) reference materials in genome in a Bottle background [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5139.

Invasive Australian populations of redlegged earth mite, Halotydeus destructor (Tucker), are evolving increasing organophosphate resistance. In addition to the canonical ace gene, the target gene of organophosphates, the H. destructor genome contains many radiated ace-like genes that vary in copy number and amino acid sequence. In this work, we characterise copy number and target-site mutation variation at the canonical ace and ace-like genes and test for potential associations with organophosphate insensitivity. This was achieved through comparisons of whole-genome pool-seq data from alive and dead mites following organophosphate exposure. A combination of increased copy number and target-site mutations at the canonical ace was associated with organophosphate insensitivity in H. destructor. Resistant populations were segregating for G119S, A201S, F331Y at the canonical ace. A subset of populations also had copy numbers of canonical ace > 2, which potentially helps overexpress proteins carrying these target-site mutations. Haplotypes possessing different copy numbers and target-site mutations of the canonical ace gene may be under selection across H. destructor populations. We also detected some evidence that increases in copy number of radiated ace-like genes are associated with organophosphate insensitivity, which might suggest potential roles in sequestration or breakdown of organophosphates. Different combinations of target-site mutations and (or) copy number variation in the canonical ace and ace-like genes may provide non-convergent ways for H. destructor to respond to organophosphate selection. However, these changes may only play a partial role in organophosphate insensitivity, which appears to have a polygenic architecture. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The aim of this study was to detect the novel copy number variation (CNV) locus of NCAM2 gene in Chinese Holstein, and to analyze the effect of the novel CNV locus in NCAM2 gene on milk composition traits. The novel CNV locus of NCAM2 gene in 310 Chinese Holstein was detected by real-time quantitative fluorescent PCR (qPCR) and association analysis was performed between the novel CNV locus in NCAM2 gene and milk composition traits in Chinese Holstein. There are three CNV types of NCAM2 gene in Chinese Holstein: gain (increased copy number), median (normal copy number) and loss (deleted copy number). Statistical analysis revealed that there was a significant association between CNV types and milk fat rate (p < 0.05). Moreover, we also discovered that the milk production and milk protein rate of gain type is higher than that of loss type, but that of mediate type is lower than that of loss type. However, in terms of somatic cell score, loss type is higher than that of gain type, but that of mediate type is lower than that of gain type. These observations suggested that gain type can be used as a candidate molecular genetic marker of milk fat rate. Highlights The CNVs of the NCAM2 gene were detected and validated in Chinese Holstein. The type of CNV was successfully implemented using qPCR. The statistical analysis indicated that the CNV of the NCAM2 gene are significantly associated with milk fat rate.

Specific copy number variants (CNVs) have been robustly associated with intellectual disability, autism, and schizophrenia. Most of the literature focus has been on documenting the existence of these phenomena. There are few data to guide therapeutic choices for these “orphan” diseases. We call for systematic and longitudinal case reports which, if carefully conducted, may provide crucial initial knowledge to guide therapeutics. We provide a step-by-step overview, a tailored set of consensus criteria for high-quality case reports, and a specific set of learning resources.

Recurrent CNVs Disrupt Three Candidate Genes in Schizophrenia Patients

Copy number variation (CNV) is implicated in important traits in multiple crop plants, but can be challenging to genotype using conventional methods. The Rhg1 locus of soybean, which confers resistance to soybean cyst nematode (SCN), is a CNV of multiple 31.2-kb genomic units each containing four genes. Reliable, high-throughput methods to quantify Rhg1 and other CNVs for selective breeding were developed. The CNV genotyping assay described here uses a homeologous gene copy within the paleopolyploid soybean genome to provide the internal control for a single-tube TaqMan copy number assay. Using this assay, CNV in breeding populations can be tracked with high precision. We also show that extensive CNV exists within Fayette, a released, inbred SCN-resistant soybean cultivar with a high copy number at Rhg1 derived from a single donor parent. Copy number at Rhg1 is therefore unstable within a released variety over a relatively small number of generations. Using this assay to select for individuals with altered copy number, plants were obtained with both increased copy number and increased SCN resistance relative to control plants. Thus, CNV genotyping technologies can be used as a new type of marker-assisted selection to select for desirable traits in breeding populations, and to control for undesirable variation within cultivars.

Copy number variation of CNVesv27061 analysis among young adults with high blood pressure using optimized droplet digital polymerase chain reaction (ddPCR) method

Copy number variation has emerged recently as an important genetic mechanism leading to phenotypic heterogeneity. The aim of our study was to determine whether copy number variants (CNVs) exist between the spontaneously hypertensive rat (SHR) and its control strain, the Wistar-Kyoto rat, whether these map to quantitative trait loci in the rat and whether CNVs associate with gene expression or blood pressure differences between the 2 strains. We performed a comparative genomic hybridization assay between SHR and Wistar-Kyoto strains using a whole-genome array. In total, 16 CNVs were identified and validated (6 because of a relative loss of copy number in the SHR and 10 because of a relative gain). CNVs were present on rat autosomes 1, 3, 4, 6, 7, 10, 14, and 17 and varied in size from 10.0 kb to 1.6 Mb. Most of these CNVs mapped to chromosomal regions within previously identified quantitative trait loci, including those for blood pressure in the SHR. Transcriptomic experiments confirmed differences in the renal expression of several genes (including Ms4a6a, Ndrg3, Egln1, Cd36, Sema3a, Ugt2b, and Idi21) located in some of the CNVs between SHR and Wistar-Kyoto rats. In F(2) animals derived from an SHRxWistar-Kyoto cross, we also found a significant increase in blood pressure associated with an increase in copy number in the Egln1 gene. Our findings suggest that CNVs may play a role in the susceptibility to hypertension and related traits in the SHR.

A new era of copy number variants (CNVs) discovery began when two separate studies, published concurrently in 2004, identified several hundred deletions and duplications in the human genome. Over the past several years, most of the CNV data were generated by microarrays. These methods have several shortcomings, such as the inability to detect copy‐neutral variants (e.g. inversions and translocations), limited sensitivity to detect smaller CNVs and poor resolution in determining CNV breakpoints especially with lower resolution microarrays. A paradigm shift in the discovery of copy‐neutral variants was attributed to the development of a sequencing‐based method known as paired‐end mapping. This method was first demonstrated to be powerful in detecting structural variants using next‐generation sequencing technologies in 2007. Further studies have also leveraged an important feature of sequencing data, where several hundred million short sequence reads are produced by next‐generation sequencers, to detect CNVs based on the abundance or density of the sequence reads aligned to a reference genome. This approach is known as depth‐of‐coverage. These emerging sequencing‐based methods will continue playing an important role in the discovery of structural variants until de novo genome assembly becomes more feasible. Key Concepts: A new era of copy number variants (CNVs) discovery began when two separate studies, published concurrently in 2004, identified several hundred deletions and duplications in the human genome. Both the sample size and the resolution of microarray are critical factors in determining the discovery of less common and smaller CNVs. ‘Human Genetic Variation’ was recognised as the ‘Breakthrough of The Year’ in 2007 by the journal Science . Other types of chromosomal rearrangements, particularly inversions and balanced translocations, have received relatively less attention. Inversions and translocations are also known as ‘copy‐neutral variants’ or ‘balanced chromosomal rearrangements’ and do not involve changes in copies number. Collectively these copy number and copy‐neutral variants are broadly classified as ‘structural variants’. The paradigm shift in the discovery of copy‐neutral variants was attributed to the development of the paired‐end mapping (PEM) method and concurrent advances in next‐generation sequencing technologies. Further studies have also leveraged on an important feature of next‐generation sequencing data where several hundred million short sequence reads are produced to detect CNVs based on the density of the sequence reads aligning to the reference genome, and this approach is known as depth‐of‐coverage (DOC). Although the PEM and DOC methods have overcome the major shortcomings of microarrays in detecting structural variants, these methods have their own weaknesses. The emerging sequencing‐based methods (PEM and DOC) will continue to play a role in the discovery of structural variants until de novo genome assembly is more feasible

You have accessJournal of UrologyCME1 Apr 2023MP04-08 COMPREHENSIVE ANALYSIS OF COPY NUMBER VARIATION AND SENSITIVITY TO TARGETED THERAPY IN RENAL CELL CARCINOMA USING IN-HOUSE CANCER GENE PANEL TESTING Akihito Takeuchi, Mayu Takeda, Eiji Sugihara, Yoshinari Muto, Sachio Nohara, Shigeki Tanishima, Kenji Zennami, Kiyoshi Takahara, Tetsuya Tsukamoto, Ryoichi Shiroki, Hideyuki Saya, and Makoto Sumitomo Akihito TakeuchiAkihito Takeuchi More articles by this author , Mayu TakedaMayu Takeda More articles by this author , Eiji SugiharaEiji Sugihara More articles by this author , Yoshinari MutoYoshinari Muto More articles by this author , Sachio NoharaSachio Nohara More articles by this author , Shigeki TanishimaShigeki Tanishima More articles by this author , Kenji ZennamiKenji Zennami More articles by this author , Kiyoshi TakaharaKiyoshi Takahara More articles by this author , Tetsuya TsukamotoTetsuya Tsukamoto More articles by this author , Ryoichi ShirokiRyoichi Shiroki More articles by this author , Hideyuki SayaHideyuki Saya More articles by this author , and Makoto SumitomoMakoto Sumitomo More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) are the main drugs used to treat metastatic renal cell carcinoma (mRCC), but there are no effective biomarkers to determine their use. In this study, we aimed to identify factors that correlate with staging of RCC and drug sensitivity to mRCC based on the results of an in-house cancer gene panel test at Fujita Health University. METHODS: 133 patients with RCC (70 non-metastatic RCC and 63 mRCC). DNA was analyzed for mutations in 143 cancer genes using the PleSSision Rapid cancer gene panel test. NextSeq2000 was used as the next-generation sequencer. From the curated reports obtained, factors including each gene mutation information and the correlation between copy number (CN) variation (CNV) cluster analysis information, clinicopathological information, and therapeutic response to ICI or TKI was examined. In this study, the therapeutic response was evaluated with RECIST1.1 and complete response and partial response were determined as objective response (OR). The endpoint was progression-free survival 2 (PFS2) defined as the interval between first-line treatment and the second relapse. RESULTS: Mutational analysis showed no difference in the pattern of genetic variation with or without metastasis. CNV analyses showed a marked trend toward a decrease in CN on homologous recombination repair genes in a part of mRCCs. Cluster analysis of CNV information for each gene in response to TKI treatment in mRCC revealed multiple regions of decreased or increased CN. The main regions of increased CN were shown to be those formed by CN gain of oncogenes on chromosomes 7 and 12. Detailed analysis of this hot spot showed that when the cutoff value of sum of CN for KRAS, ERBB3, CDK4, and MDM2 for chromosome 12 set to 9, 8 (72.7%) of 11 patients with CN 9 or higher were in OR group, while 4 (7.7%) of 52 patients with CN less than 9 were in OR group, showing a significant difference between the two groups (P < 0.001). Similarly, when the cutoff value of sum of CN for EGFR, MET, and BRAF for chromosome 7 set to 8, 5 (83.3%) of 6 patients with CN 8 or higher were in OR group, while 7 (12.3%) of 57 patients with CN < 8 were in OR group, showing a significant difference between the two groups (p<0.001). On the other hand, genetic markers predicting susceptibility to ICI could not be identified. Kaplan-Meier analysis showed that the group of patients with higher CN values for either chromosome 7 or 12 had better PFS2 after TKI treatment than the group of patients with lower CN values for either chromosome 7 or 12 (p<0.001). CONCLUSIONS: Our results from in-house cancer gene panel tests successfully showed an association between RCC progression and cancer-related gene alterations. Furthermore, the results showed the possibility of predicting TKI treatment sensitivity in mRCC by focusing on CN gain of oncogenes in specific chromosomal regions. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e36 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akihito Takeuchi More articles by this author Mayu Takeda More articles by this author Eiji Sugihara More articles by this author Yoshinari Muto More articles by this author Sachio Nohara More articles by this author Shigeki Tanishima More articles by this author Kenji Zennami More articles by this author Kiyoshi Takahara More articles by this author Tetsuya Tsukamoto More articles by this author Ryoichi Shiroki More articles by this author Hideyuki Saya More articles by this author Makoto Sumitomo More articles by this author Expand All Advertisement PDF downloadLoading ...

Purpose: Immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKIs) are the main drugs used to treat metastatic renal cell carcinoma (mRCC), but there are no effective biomarkers to determine their use. In this study, we aimed to identify factors that correlate with staging of RCC and drug sensitivity to mRCC based on the results of an in-house cancer gene panel test at Fujita Health University. Methods: 133 patients with RCC (70 non-metastatic RCC and 63 mRCC). DNA was analyzed for mutations in 143 cancer genes using the PleSSision Rapid cancer gene panel test. NextSeq2000 was used as the next-generation sequencer. From the curated reports obtained, factors including each gene mutation information and the correlation between copy number (CN) variation (CNV) cluster analysis information, clinicopathological information, and therapeutic response to ICI or TKI was examined. In this study, the therapeutic response was evaluated with RECIST1.1 and complete response and partial response were determined as objective response (OR). The endpoint was progression-free survival 2 (PFS2) defined as the interval between first-line treatment and the second relapse. Result: Mutational analysis showed no difference in the pattern of genetic variation with or without metastasis. CNV analyses showed a marked trend toward a decrease in CN on homologous recombination repair genes in a part of mRCCs. Cluster analysis of CNV information for each gene in response to TKI treatment in mRCC revealed multiple regions of decreased or increased CN. The main regions of increased CN were shown to be those formed by CN gain of oncogenes on chromosomes 7 and 12. Detailed analysis of this hot spot showed that when the cutoff value of sum of CN for KRAS, ERBB3, CDK4, and MDM2. for chromosome 12 set to 9, 8 (72.7%) of 11 patients with CN 9 or higher were in OR group, while 4 (7.7%) of 52 patients with CN less than 9 were in OR group, showing a significant difference between the two groups (P &amp;lt; 0.001). Similarly, when the cutoff value of sum of CN for EGFR, MET, and BRAF for chromosome 7 set to 8, 5 (83.3%) of 6 patients with CN 8 or higher were in OR group, while 7 (12.3%) of 57 patients with CN &amp;lt; 8 were in OR group, showing a significant difference between the two groups (P &amp;lt; 0.001). On the other hand, genetic markers predicting susceptibility to ICI could not be identified. Kaplan-Meier analysis showed that the group of patients with higher CN values for either chromosome 7 or 12 had better PFS2 after TKI treatment than the group of patients with lower CN values for either chromosome 7 or 12 (P &amp;lt; 0.001). Conclusion: Our results from in-house cancer gene panel tests successfully showed an association between RCC progression and cancer-related gene alterations. Furthermore, the results showed the possibility of predicting TKI treatment sensitivity in mRCC by focusing on CN gain of oncogenes in specific chromosomal regions. Citation Format: Akhito Takeuchi, Mayu Takeda, Eiji Sugihara, Yoshinari Muto, Sachio Nohara, Shigeki Tanishima, Kenji Zennami, Kiyoshi Takahara, Tetsuya Tsukamoto, Ryoichi Shiroki, Hideyuki Saya, Makoto Sumitomo. Comprehensive analysis of copy number variation and sensitivity to targeted therapy in renal cell carcinoma using in-house cancer gene panel testing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4331.

Copy number variation is now recognized as an important class of risk factor for several child psychiatric disorders. In this article, we first explain what copy number variants (CNVs) are. We then consider key findings and what these have told us about the etiology of these conditions. Finally, we discuss whether these findings can yet translate into clinical practice.

e16054 Background: Several studies have displayed a sustained clinical response to immune checkpoint inhibitors with profound clinical improvement in patients with microsatellite instability-high (MSI-H) metastatic colorectal cancer (CRC). MSI-H tumors may display higher numbers of TILs, many of which can be directed against tumor-related neoantigens. We set out to examine the underline mechanism in gene level to further explain the differences between MSI-H and MSS CRC. Methods: Whole exome sequencing(WES) was performed on 68 CRC patient.MSI status of these patients were determined using a targeted next generation sequencing panel covering 100 MSI loci. TCGA database was used to analyze WES data of 364 CRC patients with different MSI states, and immune signature was used to analyze the corresponding relationship of immune cell expression in different populations. Results: Of 56 MSS patients, 58.9% (33/56) had copy number variants (CNV) in DIDO1, and 1.8 (1/56) had single nucleotide variants(SNV) in DIDO1. Of 12 MSI-H patients, 33.3% (4/12) had SNV in DIDO1 gene, None had CNV in DIDO1. TCGA database showed that of 308 MSS patients, 8% (24/308) had CNV in DIDO1 and 3% (9/308) had SNV in DIDO1. Of 56 MSI-H patient, 39% (22/56) had SNV in DIDO1 and only one patent had both SNV and CNV in DIDO1. Gene variation of DIDO1 of 68 CRC patients showed a correlation with TCGA database. Gene variation of DIDO1 in MSI-H patients are mainly SNV, and the incidence of CNV is very small (4.3% of DIDO variation).While, gene variation of DIDO1 in MSS patients are mainly CNV, followed by SNV, indicating DIDO1 CNV are highly possible occurring in MSS patients. Furthermore we analyzed the relationship between the copy number of DIDO1 and immune signature through CRC TCGA database. The results showed increase of DIDO1 copy number was negatively correlated with the abundance of NK cells (P = 0.00048), T cells (P = 8.23×10-5) and macrophages (P = 1.75×10-8). Conclusions: Our results showed that different MSI status display unique patent of DIDO1 gene variation, which may be related to different immune micro environment among MSI-H/MSS patients.