Two Novel Distinct Subtypes of Myeloid Neoplasms Molecularly Associated with Histone H3K36 Methylations

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2841-2841 ◽  
Author(s):  
Yosaku Watatani ◽  
Yasunobu Nagata ◽  
Vera Grossmann ◽  
Yusuke Okuno ◽  
Tetsuichi Yoshizato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Copy Number Variations ◽  
Healthy Controls ◽  
Employment Equity ◽  
Gene Deletions ◽  
Myeloid Neoplasms ◽  
Frequent Mutations ◽  
Equity Ownership

Abstract Myelodysplastic syndromes (MDS) and related disorders are a heterogeneous group of chronic myeloid neoplasms with a high propensity to acute myeloid leukemia. A cardinal feature of MDS, as revealed by the recent genetic studies, is a high frequency of mutations and copy number variations (CNVs) affecting epigenetic regulators, such as TET2, IDH1/2, DNMT3A, ASXL1, EZH2, and other genes, underscoring a major role of deregulated epigenetic regulation in MDS pathogenesis. Meanwhile, these mutations/deletions have different impacts on the phenotype and the clinical outcome of MDS, suggesting that it should be important to understand the underlying mechanism for abnormal epigenetic regulation for better classification and management of MDS. SETD2 and ASH1L are structurally related proteins that belong to the histone methyltransferase family of proteins commonly engaged in methylation of histone H3K36. Both genes have been reported to undergo frequent somatic mutations and copy number alterations, and also show abnormal gene expression in a variety of non-hematological cancers. Moreover, germline mutation of SETD2 has been implicated in overgrowth syndromes susceptible to various cancers. However, the role of alterations in these genes has not been examined in hematological malignancies including myelodysplasia. In this study, we interrogated somatic mutations and copy number variations, among a total of 1116 cases with MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), who had been analyzed by target deep sequencing (n=944), and single nucleotide polymorphism-array karyotyping (SNP-A) (n=222). Gene expression was analyzed in MDS cases and healthy controls, using publically available gene expression datasets. SETD2 mutations were found in 6 cases, including 2 with nonsense and 4 with missense mutations, and an additional 10 cases had gene deletions spanning 1.8-176 Mb regions commonly affecting the SETD2 locus in chromosome 3p21.31, where SETD2 represented the most frequently deleted gene within the commonly deleted region. SETD2 deletion significantly correlated with reduced SETD2 expression. Moreover, MDS cases showed a significantly higher SETD2 expression than healthy controls. In total, 16 cases had either mutations or deletions of the SETD2 gene, of which 70% (7 out of 10 cases with detailed diagnostic information) were RAEB-1/2 cases. SETD2 -mutated/deleted cases had frequent mutations in TP53 (n=4), SRSF2 (n=3), and ASXL1 (n=3) and showed a significantly poor prognosis compared to those without mutations/deletions (HR=3.82, 95%CI; 1.42-10.32, P=0.004). ASH1L, on the other hand, was mutated and amplified in 7 and 13 cases, respectively, of which a single case carried both mutation and amplification with the mutated allele being selectively amplified. All the mutations were missense variants, of which 3 were clustered between S1201 and S1209. MDS cases showed significantly higher expression of ASH1L compared to healthy controls, suggesting the role of ASH1L overexpression in MDS development. Frequent mutations in TET2 (n=8) and SF3B1 (n=6) were noted among the 19 cases with ASH1L lesions. RAEB-1/2 cases were less frequent (n=11) compared to SETD2-mutated/deleted cases. ASH1L mutations did not significantly affect overall survival compared to ASH1L-intact cases. Gene Set Expression Analysis (Broad Institute) on suppressed SETD2 and accelerated ASH1L demonstrated 2 distinct expression signatures most likely due to the differentially methylated H3K36. We described recurrent mutations and CNVs affecting two histone methyltransferase genes, which are thought to represent novel driver genes in MDS involved in epigenetic regulations. Given that SETD2 overexpression and reduced ASH1L expression are found in as many as 89% of MDS cases, deregulation of both genes might play a more role than expected from the incidence of mutations and CNVs alone. Although commonly involved in histone H3K36 methylation, both methyltransferases have distinct impacts on the pathogenesis and clinical outcome of MDS in terms of the mode of genetic alterations and their functional consequences: SETD2 was frequently affected by truncating mutations and gene deletions, whereas ASH1L underwent gene amplification without no truncating mutations, suggesting different gene targets for both methyltransferases, which should be further clarified through functional studies. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Shih:Novartis: Research Funding.

scholarly journals Role of Copy Number Variations in ADHD

2020 ◽  
Author(s):  
Danijela Krgović
Keyword(s):  
Autism Spectrum Disorder ◽  
Attention Deficit ◽  
Neurodevelopmental Disorders ◽  
Copy Number ◽  
Autism Spectrum ◽  
Copy Number Variations ◽  
Spectrum Disorder ◽  
Genetic Diagnostics ◽  
Healthy Controls

Copy number variations (CNV) have an important role in etiology of neurodevelopmental disorders (NDD). Among them, individuals with attention-deficit and hyperactivity disorders (ADHD) have 1.33 times higher overall rate of CNVs larger than 100 kb compared to healthy controls. These CNVs are often shared with other NDDs and neuropsychiatric disorders such as schizophrenia (SCZ) and autism spectrum disorder (ASD), although duplications of 15q13.3 and 16p13.11 have been found enriched in ADHD cohorts. CNVs provide new opportunities for studying and management of psychiatric disorders including ADHD. Therefore this chapter provides a brief overview of the literature on this topic and presents the benefits of CNV genetic diagnostics in ADHD patients.

Recurrent Mutations of Multiple Components of Cohesin Complex in Myeloid Neoplasms

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 782-782
Author(s):  
Ayana Kon ◽  
Lee-Yung Shih ◽  
Masashi Minamino ◽  
Masashi Sanada ◽  
Yuichi Shiraishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Gene Mutations ◽  
Cohesin Complex ◽  
Wild Type ◽  
Myeloid Neoplasms ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
Equity Ownership

Abstract Abstract 782 Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in both acute and chronic myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. In the previous study, we analyzed 29 paired tumor-normal samples with chronic myeloid neoplasms with myelodysplastic features using whole exome sequencing (Yoshida et al., Nature 2011). Although the major discovery was frequent spliceosome mutations tightly associated with myelodysplasia phenotypes, hundreds of unreported gene mutations were also identified, among which we identified recurrent mutations involving STAG2, a core cohesin component, and also two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex conserved across species and is composed of four core subunits, i.e., SMC1, SMC3, RAD21 and STAG proteins, together with several regulatory proteins. Forming a ring-like structure, cohesin is engaged in cohesion of sister chromatids in mitosis, post-replicative DNA repair and regulation of gene expression. To investigate a possible role of cohesin mutations in myeloid leukemogenesis, an additional 534 primary specimens of various myeloid neoplasms was examined for mutations in a total of 9 components of the cohesin and related complexes, using high-throughput sequencing. Copy number alterations in cohesin loci were also interrogated by SNP arrays. In total, 58 mutations and 19 deletions were confirmed by Sanger sequencing in 73 out of 563 primary myeloid neoplasms (13%). Mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205) and CML (8/65), with much lower mutation frequencies in MPN (2/76), largely in a mutually exclusive manner. In MDS, mutations were more frequent in RCMD and RAEB (19.5%) but rare in RA, RARS, RCMD-RS and 5q- syndrome (3.4%). Cohesin mutations were significantly associated with poor prognosis in CMML, but not in MDS cases. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles was performed in 19 cases with cohesin mutations. Majority of the cohesin mutations (16/19) existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we investigated a possible impact of mutations on cohesin functions, where 17 myeloid leukemia cell lines with or without cohesin mutations were examined for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of one or more components of cohesin was substantially reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we examined the effect of forced expression of wild-type cohesin on cell proliferation of cohesin-defective cells. Introduction of the wild-type RAD21 and STAG2 suppressed the cell growth of RAD21- (Kasumi-1 and MOLM13) and STAG2-defective (MOLM13) cell lines, respectively, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, 23 cohesin-mutated cases of our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Alternatively, a growing body of evidence suggests that cohesin regulate gene expression, arguing for the possibility that cohesin mutations might participate in leukemogenesis through deregulated gene expression. Of additional note, the number of non-silent mutations determined by our whole exome analysis was significantly higher in 6 cohesin-mutated cases compared to non-mutated cases. Since cohesin also participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, we report a new class of common genetic targets in myeloid malignancies, the cohesin complex. Our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Frequency Of Gene Usage and Copy Number Variation Within The Rearranged Immunoglobin Heavy-Chain Variable Locus Based On Immune Repertoire Sequencing

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3486-3486
Author(s):  
Mark J. Rieder ◽  
David Williamson ◽  
Anna Sherwood ◽  
Ryan O. Emerson ◽  
Cindy Desmarais ◽  
...  
Keyword(s):  
Copy Number Variation ◽  
Copy Number ◽  
Healthy Controls ◽  
Immune Repertoire ◽  
Employment Equity ◽  
Ighv Gene ◽  
Gene Usage ◽  
Number Variation ◽  
Equity Ownership ◽  
V Gene

Abstract The human adaptive immune system is composed of both B and T cells that undergo somatic recombination at specific loci to create rearrangements of Variable (V), Diversity (D) and Joining (J) gene segments. For the B-cell immunoglobin receptor heavy-chain (IGH), the CDR3 regions are defined by the VDJ gene segments and nucleotide insertions/deletions at these junctions that create the vast sequence diversity of the IGH repertoire. Characterizing the germline DNA in these regions is impeded by the high sequence similarity between gene segments, mutation and copy-number variation (i.e. large insertions/deletions). Currently, there is a fundamental lack of information about the baseline IGH immune repertoire V gene usage and diversity within healthy human controls. To provide an estimate of this, we sequenced functionally recombined gene segments to infer the underlying gene structure. From a set of 132 healthy controls we sorted C19+/CD27+ B-cells from whole blood and amplified genomic DNA using a highly multiplexed PCR assay that targeted the rearranged IGH receptor locus. Following DNA sequencing and data processing to assign V, D and J gene families and names, we examined the usage frequency of IGHV gene segments across all individuals. We found that of the 98 V gene segments only 56 (57%) were used at a frequency > 0.1%, and ∼10 showed little to no usage (present in<1% of individuals). This data also allowed us to identify two IGHV genes currently annotated as orphons (pseudogenes assigned to an alternate chromosomal location) that had unambiguous functional usage (IGHV4/OR15-8; IGHV3/OR16-09) and therefore must reside at the IGH locus on chromosome 14. Finally, by taking this functional approach we were able to screen all V gene segments for germline copy-number variation (e.g. large insertion/deletion events encompassing individual genes) by looking for an excess of deletion events or modal changes in gene usage. We confirmed that existence of 12 of 15 previously identified deleted IGHV gene segments. Strong deletion evidence was observed for an additional six IGHV genes (IGHV3-NL1, IGHV3-33, IGHV1-24, IGHV4-04, IGHV3-41, IGHV3-35) and ten with highly likely germline deletion events. These data suggest that functional immune profiling of rearranged immune receptors provides a more robust method of identifying individual structural variation and provides insight into the immune repertoire of healthy controls. Disclosures: Rieder: Adaptive Biotechnologies: Employment, Equity Ownership. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Desmarais:Adaptive Biotechnologies: Employment, Equity Ownership. Chung:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

scholarly journals SNP Variants at 16p13.11 Clarify the Role of the NDE1/miR-484 Locus in Major Mental Illness in Finland

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Vishal Sinha ◽  
Alfredo Ortega-Alonso ◽  
Liisa Ukkola-Vuoti ◽  
Outi Linnaranta ◽  
Amanda B Zheutlin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mental Illness ◽  
Gene Expression Data ◽  
Copy Number ◽  
Copy Number Variations ◽  
Dependent Manner ◽  
Expression Data ◽  
Factor Binding Site ◽  
Association Pattern

Abstract Through copy number variations, the 16p13.11 locus has been consistently linked to mental disorders. This locus contains the NDE1 gene, which also encodes microRNA-484. Both of them have been highlighted to play a role in the etiology of mental illness. A 4-SNP haplotype spanning this locus has been shown to associate with schizophrenia in Finnish females. Here we set out to identify any functional variations implicated by this haplotype. We used a sequencing and genotyping study design to identify variations of interest in a Finnish familial cohort ascertained for schizophrenia. We identified 295 variants through sequencing, none of which were located directly within microRNA-484. Two variants were observed to associate with schizophrenia in a sex-dependent manner (females only) in the whole schizophrenia familial cohort (rs2242549 P = .00044; OR = 1.20, 95% CI 1.03–1.40; rs881803 P = .00021; OR = 1.20, 95% CI 1.02–1.40). Both variants were followed up in additional psychiatric cohorts, with neuropsychological traits, and gene expression data, in order to further examine their role. Gene expression data from the familial schizophrenia cohort demonstrated a significant association between rs881803 and 1504 probes (FDR q &lt; 0.05). These were significantly enriched for genes that are predicted miR-484 targets (n = 54; P = .000193), and with probes differentially expressed between the sexes (n = 48; P = .000187). While both SNPs are eQTLs for NDE1, rs881803 is located in a predicted transcription factor binding site. Based on its location and association pattern, we conclude that rs881803 is the prime functional candidate under this locus, affecting the roles of both NDE1 and miR-484 in psychiatric disorders.

scholarly journals MYC Rearrangements in Multiple Myeloma Are Complex, Can Involve More Than Five Different Chromosomes, and Correlate with Increased Expression of MYC and a Distinct Downstream Gene Expression Pattern

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 65-65
Author(s):  
Aneta Mikulasova ◽  
Cody Cody Ashby ◽  
Ruslana G. Tytarenko ◽  
Michael Bauer ◽  
Konstantimos Mavrommatis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Board Of Directors ◽  
Copy Number ◽  
Chromosomal Rearrangement ◽  
Hot Spot ◽  
Chromosomal Rearrangements ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Downstream Gene Expression

Abstract Introduction: The proto-oncogene MYC (locus 8q24.21) is a key transcription factor in multiple myeloma (MM) resulting in significant gene deregulation and impacting on many biological functions, including cell growth, proliferation, apoptosis, differentiation, and transformation. Chromosomal rearrangement and copy number change at the MYC locus are secondary events involved in MM progression, which are thought to lead to aggressive disease. Current analyses of the MYC locus have not been large and have reported rearrangements in 15% of new-diagnosed MM. However, more recent studies using advanced genomic techniques suggest that the frequency of MYC rearrangements may be much higher, and that a full reassessment of the role of MYC in MM pathogenesis may be critical. In this study, we analyzed 1280 MM patients to provide a better understanding of the role of this important genomic driver in MM pathogenesis. Methods: In total, 1280 tumor normal pairs of CD138 sorted bone marrow plasma cells and their germline control samples were analyzed by: 1. Targeted sequencing of 131 genes and 27 chromosome regions (n=100) with 4.5 Mb captured region surrounding MYC ; 2. Exome sequencing (n=461) with 2.3 Mb captured region surrounding MYC ; 3. Whole genome sequencing (n=719). Normalized tumor/germline depth ratio in targeted-sequencing cases and MANTA were used for detection of somatic copy number and structural variants. Expression analysis was performed using RNA-seq or microarrays. Results: MYC translocations were found in 25% (323/1280) of patients and occurred most frequently as inter-chromosomal translocations involving 2-5 chromosomes (90%, 291/323). Of the remaining cases, 5% (17/323) of the translocations involved inversion of chromosome 8 and 5% (15/323) were complex, affecting more than 5 chromosomal loci. The proportion of MYC translocations involving 2, 3, 4, and 5 loci was 62% (200/323), 23% (74/323), 8% (26/323) and 3% (8/323), respectively. Using abnormal rearranged cases (29/100), we found copy number imbalances &gt;14.2 kb in size associated with a MYC translocation in 76% (22/29). Another 7% (2/29) of cases with translocations showed complex intra-chromosomal rearrangement. A region of 2.0 Mb surrounding MYC was identified as a translocation breakpoint hot-spot incorporating 96% of breakpoints. This region also contained two hotspots for chromosomal gain and tandem duplications. MYC rearrangements were not randomly distributed across the spectrum of MM with an excess being seen in hyperdiploidy (76% of rearranged samples, P &lt;0.0001). Importantly, 67% (207/308) of cases with a MYC translocation involving 5 or less chromosomes had one of the commonly known super-enhancers involved in the translocation. Gene expression analysis was used to explore the impact of these events on downstream gene expression patterns. The results showed that inter- and/or intra-chromosomal rearrangements were associated with a significantly (P &lt;0.0001) higher MYC expression (4.1-fold). In patients where rearrangements were associated with additional copies of MYC there was higher expression of MYC in comparison to cases with a translocation but lacking copy number gain (P=0.04). To identify downstream genes deregulated by MYC rearrangements we compared gene expression between those with and without a translocation, independently of hyperdiploidy. Genes that showed &gt;2-fold change in expression (P &lt;0.01) included MYC and the non-protein coding oncogene PVT1 that is located next to MYC . Genes with significantly lower levels of expression were involved in B-cell biology including CD79A and AHR, or were associated with cell proliferation, migration, adhesion, apoptosis and/or angiogenesis (FGF16, ADAMTS1, FBXL7, HRK, PDGFD, and PRKD1) . Conclusions: This study confirms the central role of MYC in the pathogenesis of clinical cases of MM, and as such defining it as a critical therapeutic target. We will be able to target MYC better if we understand how it is deregulated and in this respect we show that the MYC locus rearrangements are complex and it is a hot-spot for heterogeneous inter- as well intra-chromosomal rearrangements, including complex rearrangements involving &gt;5 chromosomes. These events lead to increased MYC expression consistent with it being a driver of disease progression, particularly in the hyperdiploid subset of MM. Disclosures Mavrommatis: Celgene Corporation: Employment. Trotter: Celgene Corporation: Equity Ownership; Celgene Institute for Translational Research Europe: Employment. Davies: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Thakurta: Celgene Corporation: Employment, Equity Ownership. Morgan: Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria.

scholarly journals In Silico Modeling of Oncogenic Drivers in Waldenstrom Macroglobulinemia to Assess Additional Therapeutic Targets within the BCR Signaling Pathway Identifies MEK1/2 As a Target: Potential Therapeutic Role of Binimetinib

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1279-1279
Author(s):  
Aneel Paulus ◽  
Sharoon Akhtar ◽  
Shireen Vali ◽  
Ansu Kumar ◽  
Neeraj Kumar Singh ◽  
...  
Keyword(s):  
In Silico ◽  
Copy Number ◽  
Disease Stage ◽  
Advanced Stage ◽  
Employment Equity ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Bcr Signaling ◽  
Equity Ownership

Abstract Background: The observation that B-cell receptor (BCR) signaling play a critical role in the survival of malignant Waldenstrom macroglobulinemia (WM) cells is highlighted by clinical efficacy of the BTK-inhibitor, ibrutinib in WM patients. BCR signaling leads to downstream activation of several intermediary components whose activity results in increased cell proliferation. Although disruption of this axis at the proximal end (BCR) has proved successful in WM, the exact role and impact of targeting the distal intermediaries remains unknown, particularly in aggressive forms of the disease. This is important as induction of resistance to ibrutinib is clearly documented which can either be mediated through mutational changes in BTK or other members within the BCR signalosome. Using a novel simulation-based approach, we reverse-engineered the WM cell molecular architecture to uncover the role of oncogenic intermediaries distal to the BCR complex in advanced-stage WM and conducted a virtual drug-screen targeting these pathways with in vitro validation. Methods: The human WM cell line, RPCI-WM1, was used in simulation and validation experiments. RPCI-WM1 was established from a highly drug-refractory advanced disease stage patient refractory to both rituximab and bortezomib. Publically available as well as proprietary genomic and cytogenetic data was utilized for the creation of the RPCI-WM1 patient avatar, which through simulation identified the salient and prominently dysregulated cellular pathways. Importantly, illustrating these pathways highlights common convergence points on increased proliferation and viability. These convergence points were then directly and indirectly targeted by simulated testing of a library of FDA approved drugs and those impacting these dysregulated pathways were shortlisted. A standardized library of equations models all the biological reactions such as enzymatic reactions, allosteric binding and protein modulation by phosphorylation, de-phosphorylation, ubiquitination, acetylation, prenylation and others. A library of FDA approved drug agents (n~150) and those in clinical study has been developed and was simulated individually and in combination on the RPCI-WM1 (advanced stage WM patient) avatar. Results: MYD88L265P mutation, absence of CXCR4 mutations and additional chromosomal aberrations (derivatives, translocations, deletions and amplifications of chromosomes 3, 6, 9, 13, 18 and 19), which are notable features of RPCI-WM1 cells were configured and modeled in silico. The RPCI-WM1 patient avatar was predicted to have increased IRAK1/4 engagement due to MYD88 mutation and high copy number (CN) of IL18. Downregulation of DUSP1 through MYD88-mediated signaling was noted to result in high ERK activity. Increase gene copy number of both FOS and ETS1, which are downstream of ERK, were noted. Notably, FOS is a key regulator of the AP1 complex, whose activity is regulated upstream by ERK. The transcription factor, ETS1 was also predicted as over expressed and ERK-mediated phosphorylation of ETS1 regulates the activity of ETS1. The RPCI-WM1 patient avatar model also indicated high AKT due to indirect convergence of multiple aberrations. Drug screening revealed sensitivity to the MEK inhibitor, binimetinib. Although no copy number variation in MEK-ERK pathway genes were detected, per simulation, MYD88 mutation through inactivation of DUSP1 activated ERK and downstream associated survival pathways. The simulation predictions were experimentally validated. As predicted, binimetinib significantly inhibited proliferation and viability of RPCI-WM1 cells (IC50 <100nM). Conclusions: Our study demonstrates the functional role and impact of MEK1/2 - an oncogenic component distal to the BCR, and whose activity can be targeted with binimetinib to elicit a lethal effect in advanced-stage WM cells. We also show the utility of a novel technology, which is capable of integrating genome-wide molecular data points to emulate the salient genomic drivers of a given tumor cell and test its sensitivity to numerous drugs in a high-throughput manner for truly personalized therapy. Disclosures Vali: Cellworks Group, Inc.: Employment, Equity Ownership. Kumar:Cellworks Group, Inc.: Employment. Singh:Cellworks Group, Inc.: Employment. Usmani:3Cellworks Research India Limited: Employment. Grover:3Cellworks Research India Limited: Employment. Abbasi:Cellworks Group, Inc.: Employment, Equity Ownership.

scholarly journals Screening for Copy Number Variations of the 15q13.3 Hotspot in CHRNA7 Gene and Expression in Patients with Migraines

2021 ◽  
Vol 43 (2) ◽  
pp. 1090-1113
Author(s):  
Mehmet Fatih Özaltun ◽  
Sırma Geyik ◽  
Şenay Görücü Yılmaz
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Molecular Mechanisms ◽  
Cholinergic Receptor ◽  
Copy Number Variations ◽  
Healthy Controls ◽  
Standard Curve ◽  
Copy Numbers ◽  
Significant Difference ◽  
Alpha 7

Background: a migraine is a neurological disease. Copy number variation (CNV) is a phenomenon in which parts of the genome are repeated. We investigated the effects of the CNV and gene expression at the location 15q13.3 in the Cholinergic Receptor Nicotinic Alpha 7 Subunit (CHRNA7) gene, which we believe to be effective in the migraine clinic. Methods: we evaluated changes in CHRNA7 gene expression levels and CNV of 15q13.3 in patients with migraine (n = 102, with aura, n = 43; without aura, n = 59) according to healthy controls (n = 120) by q-PCR. The data obtained were analyzed against the reference telomerase reverse transcriptase (TERT) gene with the double copy number by standard curve analysis. Copy numbers were graded as a normal copy (2), gain (2>), and loss (<2). Results: we analyzed using the 2−ΔΔCT calculation method. The CHRNA7 gene was significantly downregulated in patients (p < 0.05). The analysis of CNV in the CHRNA7 gene was statistically significant in the patient group, according to healthy controls (p < 0.05). A decreased copy number indicates a dosage loss. However, no significant difference was observed among gain, normal, and loss copy numbers and expression values in patients (p > 0.05). The change in CNV was not associated with the downregulation of the CHRNA7 gene. Conclusion: Downregulation of the CHRNA7 gene may contribute to the formation of migraine by inactivation of the alpha-7 nicotinic receptor (α7nAChR). The association of CNV gains and losses with migraines will lead to better understanding of the molecular mechanisms and pathogenesis, to better define the disease, to be used as a treatment target.

scholarly journals The Biological and Clinical Implications of the Alternative Splicing Landscape of 1,258 Myeloid Neoplasm Cases

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 769-769
Author(s):  
Courtney E Hershberger ◽  
Devlin C Moyer ◽  
Wencke Walter ◽  
Stephan Hutter ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Meta Analysis ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Healthy Controls ◽  
Rna Seq ◽  
Employment Equity ◽  
Myeloid Neoplasm ◽  
Myeloid Neoplasms ◽  
Equity Ownership

NGS has led to the discovery of somatic mutations in splicing factors (SF), a group of functionally related genes previously not implicated in leukemogenesis. At least one genetic aberration in the most commonly affected 7 splicing factor genes is present in the majority of patients with MDS and related diseases (MDS/MPN and AML). The most popular and plausible hypothesis is that individual splicing mutations are associated with mis-splicing of key pathogenic genes in leukemia. However, searches for the essential mis-spliced gene or pathway in engineered cell lines and murine models have not been successful despite identification of many downstream gene targets. We have designed a strategy that overcomes pitfalls and advances results of previous attempts to identify the most essential targets. First, we have collected an expansive dataset (RNA-Seq and WGS of 1258 patient samples and 63 healthy controls) which allowed us to overcome sample size limitations and exclude cases with low tumor burdens, decreasing the analytic noise. In addition to studying the common mutant SRSF2 (n=208), SF3B1 (n=282), and U2AF1 (n=69) cases, we have also studied LOH lesions (fs, ns, deletions) in the less frequently affected splicing factors LUC7L2, DDX41, PRPF8, and ZRSR2 (n=211) (Fig.1A). Unsupervised hierarchical clustering segregated patient splicing signatures by disease type, SF mutation, and SF expression. To detect significantly dysregulated alternative splicing (AS) events, samples from each disease subtype, with mutations in SF3B1 (various), SRSF2P95, U2AF1S34, or U2AF1Q157, were compared to patients without SF mutations and also healthy controls. The disease cohorts were also stratified by LUC7L2, DDX41, PRPF8, and ZRSR2 expression levels, and the lower expression groups were compared to both the higher expression groups and healthy controls. Meta-analysis revealed over 17,000 splicing variations that were significantly dysregulated in at least one of 64 comparisons (PSI≥5%, q≤.05). Statistically significant AS events in each group were overlaid to identify commonly dysregulated AS events when compared to both the disease control and the healthy bone marrow controls (Fig.1B). We characterized AS events that were unique to the myeloid neoplasm subtypes as well as specific to genetic aberrations in SFs. We also identified genes and transcripts mis-spliced in multiple groups, suggesting a convergence of splicing factor mutations on a common target gene. The vast majority of our analysis identified alterations in isoform balance, however some splice sites that were activated only in the MDS and AML cohorts but never utilized in healthy controls. Examples of these tumor-specific splicing events are found in CERS2, which was found in a majority of patient samples, and in FMNL1, which was overwhelmingly mis-spliced in SF3B1 mutant patient samples (data not shown). We have highlighted the 52 AS events that were changed most often in comparisons against disease controls and/or healthy controls. Examples of targeted exons and introns included those in ubiquitination factors, transcription factors, DNA repair factors, and oncogenes. We classified significantly changed exons by the functional domains of the translated protein. The cohorts were then stratified according to the inclusion level of the exon or intron. The inclusion groups were compared to distinguish differences both in gene expression and in dysregulation of downstream pathways. Furthermore, the exons and introns were examined for any correlation with survival in the myeloid neoplasm subtypes. Integration of these datasets provided insights into the functional impact of AS in myeloid neoplasms, e.g., TDP1 exon 12, or exon 10b of NCOR1 inclusion both is positively correlated with expression of MYC targets and negatively associated with survival in AML patients (Fig.1C-D) In sum, we have identified strong isoform candidates for the practical study of AS driven pathogenesis, utilizing both RNA-seq and the integration of publicly available exon annotation and pathway databases. Notably, our analyses have unveiled hundreds of splicing changes dysregulated at a statistically significant level, thus warranting further assessments. This assemblage of splicing patterns found in myeloid neoplasms patients' samples is the largest in existence and should greatly advance the study of pathogenic AS. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

scholarly journals Distinct, Ethnic, Clinical, and Genetic Characteristics of Myelodysplastic Syndromes with Der(1;7)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5392-5392 ◽  
Author(s):  
Rurika Okuda ◽  
Hideki Makishima ◽  
Yasuhito Nannya ◽  
Yotaro Ochi ◽  
Tetsuichi Yoshizato ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
High Frequency ◽  
Copy Number ◽  
Research Funding ◽  
Trisomy 8 ◽  
Employment Equity ◽  
Monosomy 7 ◽  
Asian Populations ◽  
Myeloid Neoplasms ◽  
Equity Ownership

der(1;7)(q10;p10) is a recurrent chromosomal abnormality found in a wide variety of myeloid neoplasms observed in as high as 6% of myelodysplastic syndromes (MDS) in Asian populations, while rarely observed in Caucasian populations. It is thought to be generated by a recombination between two highly homologous centromere alphoid sequences which lead to an unbalanced abnormality of monosomy of 7q and trisomy of 1q. However, despite the presence of -7q, der(1;7) has been associated with a better prognosis compared to monosomy 7 or other del(7q) (-7/del(7q)). In addition to its association with +8 and del(20q), frequent RUNX1 mutations and a paucity of mutated TP53 have been reported in der(1;7) tumors, but otherwise, the molecular features of this abnormality have been poorly characterized in the literature. This is most likely because it is very rare in Caucasians, even though it represents one of the most prevalent lesions among Asian populations. The purpose of our study is to clarify the frequency and mutational landscape of der(1;7) in myeloid neoplasms on the basis of targeted-capture sequencing. A total of 1,707 MDS cases, including 944 German and 763 Japanese cases, were enrolled, from which we identified 73 (4.0%) cases with der(1;7). The prevalence was >20 times higher in Japanese (9.0%) than German (0.43%) cohorts (p<0.0001). We also identified a strong male predominance in der(1;7)-positive cases (90.4%) compared to negative cases. Also including an additional 22 cases, somatic mutations and copy number abnormalities in der(1;7) were interrogated in a total of 95 cases, which included 84 (88.4%) with MDS, 9 (9.5%) with AML, and 2 (2.1%) with MPN. Among MDS patients, 29 were low-risk, 47 were high-risk, and the rest were not specified. In mutation analysis, at least one mutation was detected in 98% of der(1;7) cases, most frequently affecting RUNX1 (42%), followed by EZH2 (26%), and ETNK1 (25%). Copy number analysis showed a high frequency of del(20q) and trisomy 8 in der(1;7) cases: 27.4% and 18.9% respectively. On the basis of mutant cell fractions, most of these mutations were present in subclones acquired within the major population harboring der(1;7). In particular, most of the EZH2 (7q35-q36) mutations were thought to be secondary events in der(1;7)-positive cases, while representing initial events acquired before UPD(7q) or -7/del(7q) in der(1;7)-negative cases. Of interest, der(1;7) was associated with a low frequency of TP53 mutations, which were seen only in 3% of cases with der(1;7), whereas highly prevalent in non-der(1;7) cases with -7/del(7q) (52%), which is concordant with a better clinical outcome was observed in der(1;7) cases compared with non-der(1;7) cases with monosomy 7 or other del(7q). Another unique feature of der(1;7) positive MDS was an extremely high frequency of RUNX1 mutations. However, the most prominent finding with secondary mutations in der(1;7) cases is the frequent hot spot mutation in ETNK1, which were originally reported in 8.8% of myeloid neoplasms with MPN features, like SETBP1 mutations. ENTK1 mutations were found in as many as 25% (23/95) of der(1;7) cases, while rarely seen in -7/del(7q) (1/89) (p<0.0001) or amp(1q) (2/68) (p=0.0001). Despite the high frequency of trisomy 8 observed in der(1;7) cases, none were associated with ETNK1 mutations. In addition, all of the RAS pathway mutations (positive in 16 cases) were observed in der(1;7) cases with wild-type ETNK1, while none were in ETNK1-mutant cases. Morphologically, these ETNK1-mutated der(1;7) cases presented with an increased eosinophil count in peripheral blood (760.9/ul vs. 78.1/ul) (p<0.001), compared to those without EKNK1 mutations, suggesting that ENTK1-mutated der(1;7) cases represent a novel disease entity within der(1;7), characterized by unique genetic features and increased eosinophils. In conclusion, der(1;7) is a genetically and clinically distinct subset of myeloid neoplasms, which showed unique features that are distinct from MDS cases in -7 and other del(7q). Especially, ETNK1 mutations subdivided cases with der(1;7) into two groups of genetically distinct subsets as shown in Figure 1. In the future, inhibition of the kinase activity in ETNK1 could be a novel therapeutic strategy in such a previously unrecognized subset as characterized by der(1;7) and eosinophilia. Figure 1 Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Baer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Atsuta:Janssen Paharmaceutical K.K.: Honoraria; Mochida Pharmaceutical Co. Ltd: Honoraria; Kyowa Kirin Co., Ltd: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Handa:Ono: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ogawa:Qiagen Corporation: Patents & Royalties; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Asahi Genomics: Equity Ownership; RegCell Corporation: Equity Ownership.

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