scholarly journals PATH-17. INTRAGENIC COPY NUMBER BREAKPOINT ANALYSIS OF METHYLATION DATA FROM CNS TUMOURS IDENTIFIES NOVEL SUBGROUP-SPECIFIC CANDIDATE FUSION GENE ENRICHMENTS

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii427-iii428
Author(s):  
Alan Mackay ◽  
Yura Grabovska ◽  
Matthew Clarke ◽  
Diana Carvalho ◽  
Sara Temelso ◽  
...  
Keyword(s):  
Copy Number ◽  
Fusion Gene ◽  
High Grade Glioma ◽  
Structural Variations ◽  
Methylation Array ◽  
Kinase Gene ◽  
Integrated Diagnosis ◽  
Cns Tumours ◽  
Breakpoint Analysis ◽  
Balanced Translocations

Abstract Methylation array-based molecular profiling has redefined the classification of brain tumours and now forms an important part of their integrated diagnosis, providing both subgroup assignment and genome wide DNA copy number profiles. These latter data can be used to identify intragenic breakpoints which are frequently associated with structural variations resulting in therapeutically targetable oncogenic fusion genes. To systematically assess the landscape of these alterations, we combined publicly available methylation datasets resulting in a total of 5660 CNS tumours, around half paediatric, and including >1000 high grade glioma and DIPG. These were analysed by standard methodology (MNP, conumee), and intragenic breakpoint enrichment was compared within methylation subgroups, superfamilies, and tumours with no high-scoring classification. Benchmarking included sequence-verified cases such as infant hemispheric gliomas (IHG) with ALK(15%) and ROS1(7%) fusions, and pathognomic alterations associated with specific entities such as RELA-EPN, MYB-LGG and HGNET-MN1. We identified previously unreported enrichments of well-recognised fusion targets such as NTRK2in GBM_MID and NTRK3in DMG_K27 (both 5%), METin A_IDH / A_IDH_HG (3–5%), and FGFR1/3in GBM_G34 (8–9%). Novel recurrent kinase gene candidates to be verified and explored further include IGF1Rin 2–12% cases spanning glioma subgroups, and TIE1in poorly classified tumours. This latter ‘NOS’ group were also enriched in various transcription factor targets of breakpoints, including TCF4and PLAGL2. Despite limitations due to sample quality, resolution or balanced translocations, breakpoint analysis of methylation copy number profiles provides simple screening for structural rearrangements which may directly influence targeted therapy in paediatric CNS tumours.

scholarly journals Rapid-CNS2: Rapid comprehensive adaptive nanopore-sequencing of CNS tumors, a proof of concept study

2021 ◽  
Author(s):  
Areeba J Patel ◽  
Helin Dogan ◽  
Alexander Payne ◽  
Philipp Sievers ◽  
Natalie Schoebe ◽  
...  
Keyword(s):  
Random Forest ◽  
Copy Number ◽  
Turnaround Time ◽  
Random Forest Classifier ◽  
Diffuse Glioma ◽  
Nanopore Sequencing ◽  
Proof Of Concept ◽  
Methylation Array ◽  
Concept Study ◽  
Integrated Diagnosis

Background: The 2021 WHO classification of central nervous system tumors includes multiple molecular markers and patterns that are recommended for routine diagnostic use in addition to histology. Sequencing infrastructures for complete molecular profiling require considerable investment, while batching samples for sequencing and methylation profiling can delay turnaround time. We introduce RAPID-CNS2, a nanopore adaptive sequencing pipeline that enables comprehensive mutational, methylation and copy number profiling of CNS tumours with a single, cost-effective sequencing assay. It can be run for single samples and offers highly flexible target selection that can be personalized per case with no additional library preparation. Methods: Utilizing ReadFish, a toolkit enabling targeted nanopore sequencing without the need for library enrichment, we sequenced DNA from 22 diffuse glioma samples on a MinION device. Target regions comprised our Heidelberg brain tumor NGS panel and pre-selected CpG sites for methylation classification using an adapted random forest classifier. Pathognomonic alterations, copy number profiles, and methylation classes were called using a custom bioinformatics pipeline. The resulting data were compared to their corresponding standard NGS panel sequencing and EPIC methylation array results. Results: Complete concordance with the EPIC array was found for copy number profiles. The vast majority (94%) of pathognomonic mutations were congruent with standard NGS panel-seq data. MGMT promoter status was correctly identified in all samples. Methylation families from the random forest classifier were detected with 96% congruence. Among the alterations decisive for rendering a WHO 2021 classification-compatible integrated diagnosis, 97% of the alterations were consistent over the entire cohort (completely congruent in 19/22 cases and sufficient for unequivocal diagnosis in all 22 samples). Conclusions: RAPID-CNS2 provides a swift and highly flexible alternative to conventional NGS and array-based methods for SNV/InDel analysis, detection of copy number alterations, target gene methylation analysis (e.g. MGMT) and methylation-based classification. The turnaround time of ~5 days for this proof-of-concept study can be further shortened to < 24h by optimizing target sizes and enabling real-time computational analysis. Expected advances in nanopore sequencing and analysis hardware make the prospect of integrative molecular diagnosis in an intra-operative setting a feasible prospect in future. This low-capital approach would be cost-efficient for low throughput settings or in locations with less sophisticated laboratory infrastructure, and invaluable in cases requiring immediate diagnoses.

scholarly journals HiCancer: accurate and complete cancer genome phasing with Hi-C reads

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Weihua Pan ◽  
Desheng Gong ◽  
Da Sun ◽  
Haohui Luo
Keyword(s):  
Copy Number ◽  
Somatic Mutations ◽  
Copy Number Variations ◽  
Cancer Genome ◽  
Structural Variations ◽  
Genome Map ◽  
Linkage Information ◽  
Suboptimal Solution ◽  
Allelic Copy Number ◽  
Very High

AbstractDue to the high complexity of cancer genome, it is too difficult to generate complete cancer genome map which contains the sequence of every DNA molecule until now. Nevertheless, phasing each chromosome in cancer genome into two haplotypes according to germline mutations provides a suboptimal solution to understand cancer genome. However, phasing cancer genome is also a challenging problem, due to the limit in experimental and computational technologies. Hi-C data is widely used in phasing in recent years due to its long-range linkage information and provides an opportunity for solving the problem of phasing cancer genome. The existing Hi-C based phasing methods can not be applied to cancer genome directly, because the somatic mutations in cancer genome such as somatic SNPs, copy number variations and structural variations greatly reduce the correctness and completeness. Here, we propose a new Hi-C based pipeline for phasing cancer genome called HiCancer. HiCancer solves different kinds of somatic mutations and variations, and take advantage of allelic copy number imbalance and linkage disequilibrium to improve the correctness and completeness of phasing. According to our experiments in K562 and KBM-7 cell lines, HiCancer is able to generate very high-quality chromosome-level haplotypes for cancer genome with only Hi-C data.

scholarly journals Genomic alterations caused by HPV integration in a cohort of Chinese endocervical adenocarcinomas

2021 ◽  
Author(s):  
Wenhui Li ◽  
Wanjun Lei ◽  
Xiaopei Chao ◽  
Xiaochen Song ◽  
Yalan Bi ◽  
...  
Keyword(s):  
Copy Number ◽  
Somatic Mutations ◽  
Hpv Infection ◽  
Copy Number Variations ◽  
Cervical Adenocarcinoma ◽  
Structural Variations ◽  
Driver Genes ◽  
Genetic Changes ◽  
Genomic Changes ◽  
Whole Exome

AbstractThe association between human papillomavirus (HPV) integration and relevant genomic changes in uterine cervical adenocarcinoma is poorly understood. This study is to depict the genomic mutational landscape in a cohort of 20 patients. HPV+ and HPV− groups were defined as patients with and without HPV integration in the host genome. The genetic changes between these two groups were described and compared by whole-genome sequencing (WGS) and whole-exome sequencing (WES). WGS identified 2916 copy number variations and 743 structural variations. WES identified 6113 somatic mutations, with a mutational burden of 2.4 mutations/Mb. Six genes were predicted as driver genes: PIK3CA, KRAS, TRAPPC12, NDN, GOLGA6L4 and BAIAP3. PIK3CA, NDN, GOLGA6L4, and BAIAP3 were recognized as significantly mutated genes (SMGs). HPV was detected in 95% (19/20) of patients with cervical adenocarcinoma, 7 of whom (36.8%) had HPV integration (HPV+ group). In total, 1036 genes with somatic mutations were confirmed in the HPV+ group, while 289 genes with somatic mutations were confirmed in the group without HPV integration (HPV− group); only 2.1% were shared between the two groups. In the HPV+ group, GOLGA6L4 and BAIAP3 were confirmed as SMGs, while PIK3CA, NDN, KRAS, FUT1, and GOLGA6L64 were identified in the HPV− group. ZDHHC3, PKD1P1, and TGIF2 showed copy number amplifications after HPV integration. In addition, the HPV+ group had significantly more neoantigens. HPV integration rather than HPV infection results in different genomic changes in cervical adenocarcinoma.

scholarly journals MBCL-08. INTEGRATIVE MOLECULAR ANALYSIS OF PATIENT-MATCHED DIAGNOSTIC AND RELAPSED MEDULLOBLASTOMAS

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii389-iii389
Author(s):  
Rahul Kumar ◽  
Maximilian Deng ◽  
Kyle Smith ◽  
Anthony Liu ◽  
Girish Dhall ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Copy Number ◽  
Driver Mutations ◽  
P Value ◽  
Next Generation ◽  
Driver Genes ◽  
Methylation Array ◽  
Primary Tumors ◽  
Group 4 ◽  
Group 3

Abstract INTRODUCTION The next generation of clinical trials for relapsed medulloblastoma demands a thorough understanding of the clinical behavior of relapsed tumors as well as the molecular relationship to their diagnostic counterparts. METHODS A multi-institutional molecular cohort of patient-matched (n=126 patients) diagnostic MBs and relapses/subsequent malignancies was profiled by DNA methylation array. Entity, subgroup classification, and genome-wide copy-number aberrations were assigned while parallel next-generation (whole-exome or targeted panel) sequencing on the majority of the cohort facilitated inference of somatic driver mutations. RESULTS Comprised of WNT (2%), SHH (41%), Group 3 (18%), Group 4 (39%), primary tumors retained subgroup affiliation at relapse with the notable exception of 10% of cases. The majority (8/13) of discrepant classifications were determined to be secondary glioblastomas. Additionally, rare (n=3) subgroup-switching events of Group 4 primary tumors to Group 3 relapses were identified coincident with MYC/MYCN pathway alterations. Amongst truly relapsing MBs, copy-number analyses suggest somatic clonal divergence between primary MBs and their respective relapses with Group 3 (55% of alterations shared) and Group 4 tumors (63% alterations shared) sharing a larger proportion of cytogenetic alterations compared to SHH tumors (42% alterations shared; Chi-square p-value &lt; 0.001). Subgroup- and gene-specific patterns of conservation and divergence amongst putative driver genes were also observed. CONCLUSION Integrated molecular analysis of relapsed MB discloses potential mechanisms underlying treatment failure and disease recurrence while motivating rational implementation of relapse-specific therapies. The degree of genetic divergence between primary and relapsed MBs varied by subgroup but suggested considerably higher conservation than prior estimates.

scholarly journals The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (6) ◽  
pp. 2994-3004 ◽  
Author(s):  
M Kaouass ◽  
M Audette ◽  
D Ramotar ◽  
S Verma ◽  
D De Montigny ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Fusion Gene ◽  
Transport Systems ◽  
Coding Region ◽  
Kinase Gene ◽  
Lower Affinity ◽  
High Affinity ◽  
Polyamine Transport ◽  
Exogenous Spermidine

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.

Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene

1991 ◽  
Vol 11 (6) ◽  
pp. 3374-3378 ◽  
Author(s):  
S D Lupton ◽  
L L Brunton ◽  
V A Kalberg ◽  
R W Overell
Keyword(s):  
Thymidine Kinase ◽  
Negative Selection ◽  
Fusion Gene ◽  
Hygromycin B ◽  
Thymidine Kinase Gene ◽  
Kinase Gene ◽  
Hygromycin Phosphotransferase ◽  
Simplex Virus ◽  
Genetic Entity

The hygromycin phosphotransferase gene was fused in-frame with the herpes simplex virus type 1 thymidine kinase gene. The resulting fusion gene (termed HyTK) confers hygromycin B resistance for dominant positive selection and ganciclovir sensitivity for negative selection and provides a means by which these selectable phenotypes may be expressed and regulated as a single genetic entity.

Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences

1986 ◽  
Vol 6 (12) ◽  
pp. 4335-4343
Author(s):  
J E Ogden ◽  
C Stanway ◽  
S Kim ◽  
J Mellor ◽  
A J Kingsman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Fusion Gene ◽  
Phosphoglycerate Kinase ◽  
Human Interferon ◽  
Coding Region ◽  
Kinase Gene ◽  
Upstream Activation Sequence ◽  
Efficient Expression ◽  
Activation Sequence

The Saccharomyces cerevisiae PGK (phosphoglycerate kinase) gene encodes one of the most abundant mRNA and protein species in the cell. To identify the promoter sequences required for the efficient expression of PGK, we undertook a detailed internal deletion analysis of the 5' noncoding region of the gene. Our analysis revealed that PGK has an upstream activation sequence (UASPGK) located between 402 and 479 nucleotides upstream from the initiating ATG sequence which is required for full transcriptional activity. Deletion of this sequence caused a marked reduction in the levels of PGK transcription. We showed that PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation. We also showed that the UASPGK functions as efficiently when in the inverted orientation and that it can enhance transcription when placed upstream of a TRP1-IFN fusion gene comprising the promoter of TRP1 fused to the coding region of human interferon alpha-2.

Transcriptional insulation of the human keratin 18 gene in transgenic mice

1993 ◽  
Vol 13 (4) ◽  
pp. 2214-2223
Author(s):  
N Neznanov ◽  
I S Thorey ◽  
G Ceceña ◽  
R G Oshima
Keyword(s):  
Transgenic Mice ◽  
Thymidine Kinase ◽  
Copy Number ◽  
Thymidine Kinase Gene ◽  
Flanking Sequence ◽  
Kinase Gene ◽  
Tissue Specific ◽  
Dependent Behavior ◽  
Flanking Sequences ◽  
Keratin 18

Expression of the 10-kb human keratin 18 (K18) gene in transgenic mice results in efficient and appropriate tissue-specific expression in a variety of internal epithelial organs, including liver, lung, intestine, kidney, and the ependymal epithelium of brain, but not in spleen, heart, or skeletal muscle. Expression at the RNA level is directly proportional to the number of integrated K18 transgenes. These results indicate that the K18 gene is able to insulate itself both from the commonly observed cis-acting effects of the sites of integration and from the potential complications of duplicated copies of the gene arranged in head-to-tail fashion. To begin to identify the K18 gene sequences responsible for this property of transcriptional insulation, additional transgenic mouse lines containing deletions of either the 5' or 3' distal end of the K18 gene have been characterized. Deletion of 1.5 kb of the distal 5' flanking sequence has no effect upon either the tissue specificity or the copy number-dependent behavior of the transgene. In contrast, deletion of the 3.5-kb 3' flanking sequence of the gene results in the loss of the copy number-dependent behavior of the gene in liver and intestine. However, expression in kidney, lung, and brain remains efficient and copy number dependent in these transgenic mice. Furthermore, herpes simplex virus thymidine kinase gene expression is copy number dependent in transgenic mice when the gene is located between the distal 5'- and 3'-flanking sequences of the K18 gene. Each adult transgenic male expressed the thymidine kinase gene in testes and brain and proportionally to the number of integrated transgenes. We conclude that the characteristic of copy number-dependent expression of the K18 gene is tissue specific because the sequence requirements for transcriptional insulation in adult liver and intestine are different from those for lung and kidney. In addition, the behavior of the transgenic thymidine kinase gene in testes and brain suggests that the property of transcriptional insulation of the K18 gene may be conferred by the distal flanking sequences of the K18 gene and, additionally, may function for other genes.

scholarly journals Failure to Confirm CNVs as of Aetiological Significance in Twin Pairs Discordant for Schizophrenia

2010 ◽  
Vol 13 (5) ◽  
pp. 455-460 ◽  
Author(s):  
Shinji Ono ◽  
Akira Imamura ◽  
Shinya Tasaki ◽  
Naohiro Kurotaki ◽  
Hiroki Ozawa ◽  
...  
Keyword(s):  
Copy Number ◽  
Developmental Process ◽  
Single Nucleotide Polymorphism Array ◽  
Monozygotic Twins ◽  
Peripheral Blood Lymphocytes ◽  
Copy Number Variations ◽  
Genomic Diversity ◽  
Clinical Population ◽  
Small Subset ◽  
Structural Variations

Copy number variations (CNVs) are common structural variations in the human genome that strongly affect genomic diversity and can play a role in the development of several diseases, including neurodevelopmental disorders. Recent reports indicate that monozygotic twins can show differential CNV profiles. We searched CNVs in monozygotic twins discordant for schizophrenia to identify susceptible loci for schizophrenia. Three pairs of monozygotic twins discordant for schizophrenia were subjected to analysis. Genomic DNA samples were extracted from peripheral blood lymphocytes. We adopted the Affymetrix Genome-Wide Human SNP (Single Nucleotide Polymorphism) Array 6.0 to detect copy number discordance using Partek Genomics Suite 6.5 beta. In three twin pairs, however, validations by quantitative PCR and DNA sequencing revealed that none of the regions had any discordance between the three twin pairs. Our results support the hypothesis that epigenetic changes or fluctuation in developmental process triggered by environmental factors mainly contribute to the pathogenesis of schizophrenia. Schizophrenia caused by strong genetics factors including copy number alteration or gene mutation may be a small subset of the clinical population.

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