GLORIA

GEOMAR Library Ocean Research Information Access

X

Your email was sent successfully. Check your inbox.

An error occurred while sending the email. Please try again.

Proceed reservation?

Export
  • 1
    Online Resource
    Online Resource
    In Analogy to AML, MDS Can be Sub-Classified By Ancestral Mutations
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 823-823
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 823-823
    Abstract: Somatic mutations constitute key pathogenetic elements in MDS. Unbiased whole exome sequencing (WES) and deep NGS led to discovery of new somatic mutations and also to the recognition of i) tremendous diversity of mutations and their combinations; ii) individual intra-tumor heterogeneity and clonal hierarchy. Chromosomal lesions further increase the complexity of molecular defects. While in MDS molecular defects are acquired in order, observations made in AML highlight the importance of ancestral events; e.g., t(8;21), inv16 or t(15;17) and other lesions that are used as the basis for nosological sub-classification. Thus, it is the identity of individual ancestral events or their classes rather than the spectrum of secondary events or the distribution of mutations, that will allow for molecular, functionally-relevant and diagnostically useful classification within MDS. This would explain why only a few somatic mutations have been found to be prognostically important, as their position in the clonal hierarchy has not been accounted for. With this in mind, we applied WES (N=206) and targeted deep NGS (N=836) and studied 100 samples serially with analyses focused on ancestral events. Globally, through WES we identified and validated 2386 mutational events in 1458 genes. Of these, 112 genes were mutated at significant frequencies (q 〈 0.05); groups of affected genes involved in splicing, transcription, DNA methylation, histone modification, and others were distinguished. On average, 9 somatic events per MDS case, 10.7 in secondary AML, and 12.5 in MDS/MPN were found. Resequencing in combination with SNP-array karyotyping provided information on variant allelic frequency (VAF) adjusted for corresponding zygosity of mutations; 99% of cases displayed clear intra-tumor heterogeneity due to multiple clones defined by hierarchically acquired somatic mutational patterns. Using cross-sectional analyses, the highest mean VAF could be interpreted as consistent with the ancestral nature of the mutations, as seen for instance in a proportion of TET2 and SF3B1 mutant cases. In contrast, the lowest mean VAF indicated secondary events, as occur in NPM1 and RAS pathway mutations. Similar conclusions were made based on cross-sectional analyses showing a similar distribution of ancestral but not secondary events in MDS and sAML. All gene mutations were categorized into those that are predominantly ancestral and those that are facultatively secondary. The most frequent founder mutations were identified (TET2, DNMT3A, SF3B1, ASXL1, TP53, U2AF1, RUNX1, SRSF2) and used to sub-classify approximately 80% of patients, with the remainder containing more infrequent ancestral mutations. While in a combined fashion (as both founder and secondary events) many of these mutations were not predictive of prognosis, they gained relevance when only cases affected by ancestral mutations were used for prognostication. Thus some of the mutations, when present as secondary events may not be predictive. Founding mutations may determine subsequent clinical and molecular features. While other frequently affected genes, SF3B1 or ASXL1, are not associated with a significant increase in the number of concomitant mutations, cases with TET2 mutations showed significantly more frequent mutations per case than those with wild-type TET2 (14.6 vs. 9.1; p=0.001). Moreover, ancestral TET2 mutations were associated with concomitant mutations due to high C-to-T transitions, possibly because reduced 5-hydroxymethylcytosine might create the specific mutator milieu. Most important is the association not of any type, but of ancestral mutations with certain pathomorphologic features and outcomes. Founding TET2 mutations are associated with MPN/MDS while secondary TET2 mutations are present in MDS. Ancestral DNMT3A mutations determine a rapid progression to AML, whereas subclonal DNMT3A mutations are also found in high-risk MDS. RAS pathway mutations are ancestral in CMML and also secondarily positive in the late stage of MDS (sAML). Specific ancestral events may determine subsequent mutational events, and while both types of mutation may affect the clinical phenotype, the initial events are less diverse and more subtype-specific. In conclusion, WES clarified the distinct landscape and ordering of the somatic mutational spectrum in MDS. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.823.823
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 2
    Online Resource
    Online Resource
    Recurrent Mutations of Multiple Components of Cohesin Complex in Myeloid Neoplasms
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 782-782
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 782-782
    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.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.782.782
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 3
    Online Resource
    Online Resource
    Abstract 5117: Mutations of cohesin genes in myeloid malignancy
    American Association for Cancer Research (AACR) ; 2012
    In:  Cancer Research Vol. 72, No. 8_Supplement ( 2012-04-15), p. 5117-5117
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 72, No. 8_Supplement ( 2012-04-15), p. 5117-5117
    Abstract: Myelodysplastic syndromes and related myeloid neoplasms (myelodysplasia) are a heterogeneous group of clonal disorders showing deregulated blood cell production and a predisposition to acute myeloid leukemia, whose pathogenesis is only incompletely understood. So, to clarify the molecular pathogenesis of myelodysplasia, we performed whole-exome sequencing of paired tumor/control DNA from 29 patients with myelodysplasia, leading to the identification of novel pathway mutations of the splicing machinery in myelodysplasia (Yoshida et al., Nature, 2011). In addition to these pathway mutations, we also identified a number of previously unreported gene mutations. Among these are a missense and a nonsense mutation involving two cohesin components, STAG1 and STAG2 found in single cases, respectively. Cohesin is a multimeric protein complex and enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together during mitosis. To extend the findings in the whole-exome sequencing, we investigated mutations of cohesin complex, including STAG2/STAG1, SMC1A, SMC3 and RAD21, in 370 cases of myeloid malignancy by deep sequencing of pooled DNA. In total, 38 mutations were identified in 36 out of the 370 cases, where STAG2 and RAD21 accounted for most of the mutations. These mutations occurred in a completely mutually exclusive manner, suggesting a common impact of these mutations on the pathogenesis of myeloid neoplasms. Most mutations of STAG2 and RAD21 were nonsense or frameshift changes, or splice site mutations and widely distributed along the entire coding region, causing loss-of-function of the proteins. On the other hand, all mutations detected in SMC1A, SMC3, and STAG1 were missense changes, indicating that their functions are essential for tumor survival, complete loss of functions of which could lead to cell death. In cytogenetics, 11 cohesin-mutated cases had normal karyotypes, and only 16 out of the 36 tumors with cohesion mutations showed abnormal karyotypes, where most cases had near-diploid with only 2 patients having complex karyotypes. So far, several lines of evidence suggest that cohesin plays an important role for genomic stability and mutational inactivation of STAG2 was shown to cause aneuploidy in human cells. However, our results raise the possibility that alterations of cohesin genes could be involved in carcinogenesis at least partly through mechanisms other than causing aneuploidy. In this context, it is of note that growing evidence have shown that cohesin forms long-range chromosomal interactions and regulate gene expression in association with CTCF, mediator, or transcription factors. Further functional study should be warranted to gain new insights into the role of cohesin in the pathogenesis of myeloid malignancies as well as other human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5117. doi:1538-7445.AM2012-5117
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2012-5117
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2012
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 4
    Online Resource
    Online Resource
    Frequent pathway mutations of splicing machinery in myelodysplasia
    Springer Science and Business Media LLC ; 2011
    In:  Nature Vol. 478, No. 7367 ( 2011-10), p. 64-69
    Details
    In: Nature, Springer Science and Business Media LLC, Vol. 478, No. 7367 ( 2011-10), p. 64-69
    Type of Medium: Online Resource
    ISSN: 0028-0836 , 1476-4687
    URL: Article
    DOI: 10.1038/nature10496
    RVK:
    TA 1000
    RVK:
    UA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2011
    detail.hit.zdb_id: 120714-3
    detail.hit.zdb_id: 1413423-8
    SSG: 11
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 5
    Online Resource
    Online Resource
    Abstract 4602: Recurrent pathway mutations of multiple components of cohesin complex in myeloid neoplasms.
    American Association for Cancer Research (AACR) ; 2013
    In:  Cancer Research Vol. 73, No. 8_Supplement ( 2013-04-15), p. 4602-4602
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 73, No. 8_Supplement ( 2013-04-15), p. 4602-4602
    Abstract: 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 myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. So, we analyzed 50 paired tumor-normal samples of myeloid neoplasms using whole exome sequencing, among which we identified recurrent mutations involving STAG2, a core cohesin component, and two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex which is composed of four core subunits (SMC1, SMC3, RAD21 and STAG proteins), and is engaged in cohesion of sister chromatids, DNA repair and transcriptional regulation. To extend the findings in the whole-exome analysis, an additional 534 primary samples of various myeloid neoplasms was examined for mutations and deletions in a total of 9 components of the cohesin complexes, using high-throughput sequencing and SNP arrays. In total, mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205), in a mutually exclusive manner. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles revealed that majority of the cohesin mutations existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we examined several myeloid leukemia cell lines with or without cohesin mutations for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of several components of cohesin was significantly reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we introduced the wild-type RAD21 allele into RAD21-mutated cell lines (Kasumi-1), which effectively suppressed the proliferation of Kasumi-1, 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, about half of cohesin-mutated cases in our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Of note, the number of mutations determined by our whole exome analysis was significantly higher in cohesin-mutated cases compared to non-mutated cases. Since cohesin participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Citation Format: Ayana Kon, Lee-Yung Shih, Masashi Minamino, Masashi Sanada, Yuichi Shiraishi, Yasunobu Nagata, Kenichi Yoshida, Yusuke Okuno, Masashige Bando, Shunpei Ishikawa, Aiko Sato-Otsubo, Genta Nagae, Aiko Nishimoto, Claudia Haferlach, Daniel Nowak, Yusuke Sato, Tamara Alpermann, Teppei Shimamura, Hiroko Tanaka, Kenichi Chiba, Ryo Yamamoto, Tomoyuki Yamaguchi, Makoto Otsu, Naoshi Obara, Mamiko Sakata-Yanagimoto, Tsuyoshi Nakamaki, Ken Ishiyama, Florian Nolte, Wolf-Karsten Hofmann, Shuichi Miyawaki, Shigeru Chiba, Hiraku Mori, Hiromitsu Nakauchi, H. Phillip Koeffler, Hiroyuki Aburatani, Torsten Haferlach, Katsuhiko Shirahige, Satoru Miyano, Seishi Ogawa. Recurrent pathway mutations of multiple components of cohesin complex in myeloid neoplasms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4602. doi:10.1158/1538-7445.AM2013-4602
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2013-4602
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2013
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 6
    Online Resource
    Online Resource
    Abstract 5119: Frequent splicing pathway mutations and aberrant RNA splicing in myelodysplasia
    American Association for Cancer Research (AACR) ; 2012
    In:  Cancer Research Vol. 72, No. 8_Supplement ( 2012-04-15), p. 5119-5119
    Details
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 72, No. 8_Supplement ( 2012-04-15), p. 5119-5119
    Abstract: MDS are a group of myeloid neoplasms characterized by deregulated blood cell production and a high propensity to AML. Although a number of gene alterations have been implicated in the pathogenesis of MDS, they do not fully explain the pathogenesis of MDS. So, in order to clarify a comprehensive registry of gene mutations in MDS, we performed whole-exome sequencing of 29 cases with MDS and related myeloid neoplasm. A total of 268 somatic mutations or 9.2 mutations per sample were identified. Among these 9 genes were mutated in more than 2 cases, which not only included a spectrum of known gene targets in MDS, but also affected previously unknown genes that are commonly involved in RNA splicing pathway, including U2AF35, SRSF2 and ZRSR2. Together with additional three (SF3A1, SF3B1 and PRPF40B) found in single cases, 16 (55.2%) of the 29 discovery cases carried a mutation affecting the component of the splicing machinery. To confirm the observation, we examined 9 spliceosome genes for mutations in a large set of myeloid neoplasms. In total, 219 mutations were identified in 209 out of the 582 samples of myeloid neoplasms. RNA splicing pathway mutations were highly specific to myelodysplasia, including 19 of 23 (83%) cases with RARS, 43 of 50 (86%) RCMD-RS, 68 of 155 (44%) other MDS, 48 of 88 (55%) CMML, and 16 of 62 (26%) secondary AML with MDS features with a string preference of SF3B1 mutations to RARS and RCMD-RS and of SRSF2 to CMML, while they were rare in cases with de novo AML and MPN. Significantly, these mutations occurred in an almost completely mutually exclusive manner among mutated cases, suggesting the importance of deregulated RNA splicing in the pathogenesis of MDS. RNA splicing plays critical roles in differentiation, development, and disease and is a major source for protein diversity in higher eukaryotes. Splicing pathway mutations in myelodysplasia commonly affected those components of the splicing complex that are engaged in the 3′ splice site recognition, strongly indicating production of unspliced or aberrantly spliced RNA species are incriminated for the pathogenesis of MDS. So, to clarify the effect of these splicing mutations on RNA splicing, we expressed the wild-type and the mutant U2AF35 or SRSF2 in HeLa cells and performed whole transcriptome analysis in these cells. The results of exon array showed that the wild-type U2AF35 promoted RNA splicing correctly, whereas the mutant U2AF35 inhibited this processes and rendered intronic sequences to remain unspliced. RNA sequencing additionally showed that the number of reads that encompassed the exon/intron junctions was significantly increased in mutant U2AF35-transduced cells. This result means that mutant U2AF35 actually induced impaired 3′-splice site recognition during pre-mRNA processing. In conclusion, our study demonstrated that abnormal RNA splicing caused by mutations of multiple genes on RNA splicing pathway is a common feature of myelodysplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5119. doi:1538-7445.AM2012-5119
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
    URL: Article
    DOI: 10.1158/1538-7445.AM2012-5119
    RVK:
    XA 36000
    RVK:
    XA 10000
    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2012
    detail.hit.zdb_id: 2036785-5
    detail.hit.zdb_id: 1432-1
    detail.hit.zdb_id: 410466-3
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 7
    Online Resource
    Online Resource
    Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms
    Springer Science and Business Media LLC ; 2013
    In:  Nature Genetics Vol. 45, No. 10 ( 2013-10), p. 1232-1237
    Details
    In: Nature Genetics, Springer Science and Business Media LLC, Vol. 45, No. 10 ( 2013-10), p. 1232-1237
    Type of Medium: Online Resource
    ISSN: 1061-4036 , 1546-1718
    URL: Article
    DOI: 10.1038/ng.2731
    RVK:
    XA 10000
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2013
    detail.hit.zdb_id: 1494946-5
    SSG: 12
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 8
    Online Resource
    Online Resource
    Clinical “MUTATOME” Of Myelodysplastic Syndrome; Comparison To Primary Acute Myelogenous Leukemia
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 518-518
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 518-518
    Abstract: Chromosomal aberrations and somatic mutations constitute key elements of the pathogenesis of myelodysplastic syndromes (MDS), a clonal hematologic malignancy characterized by cytopenias, a dysplastic bone marrow and propensity to clonal evolution. Next generation sequencing (NGS) enables definition of somatic mutational patterns and clonal architecture as a discovery platform, and for clinical applications. We systematically applied NGS to 707 cases of MDS and MDS-related disorders. 205 cases (low-risk MDS: N=78, high-risk MDS: N=42, MDS/MPN: N=48 and sAML: N=37) were tested by whole exome sequencing (WES). For validation in an additional 502 patients (low-risk MDS: N=192, high-risk MDS: N=104, MDS/MPN: N=111 and sAML: N=95), targeted deep NGS was applied for 60 index genes which were most commonly affected in the cohort analyzed by WES. For NGS data analysis a statistical pipeline was developed to focus on: i) identification of the most relevant somatic mutations, and ii) minimization of false positive results. We studied serial samples from 21 exemplary informative patients. We also compared somatic mutational patterns to those seen in primary AML TCGA cohort (N=201). Given the size of the cohort, there was, for example, a 87% chance of seeing mutations at a frequency of 1% and a 98% of seeing those with a frequency of 2%. While focusing on the most common events, we observed 1117 somatic mutations in 199 genes. The 88 genes mutated mutated in 〉 1% of cases with MDS carried 388 mutations in MDS+sAML (2.5/case), 128 in MDS/MPN (2.7/case) and 398 in pAML (2.0/case). The average number of mutations per case increased during progression (2.2 in lower-risk, 2.8 in higher-risk MDS, 3.4 in sAML). In MDS, the 30 most frequently affected genes were present at least once in 70% of patients. The 30 most frequently mutated genes in MDS/MPN were mutated in 82% of patients. Individual mutations were also sub-grouped according to their function. When we compared three MDS subcategories (lower-risk, higher-risk MDS and sAML) in a cross-sectional view, RTK family, RAS family, IDH family and cohesin family mutations were more frequently detected in the sAML group than in the MDS group. In contrast, the frequency of the DNMT family, TET2 and ASXL family gene mutations did not increase in frequency in the sAML cohort. In addition to better definition of mutational patterns of known genes, we have also defined new mutations, including in the RNA helicase family and the BRCC3pathway. Clonal architecture analysis indicates that mutations of TET2, DNMT3A, ASXL1, and U2AF1 most likely represent ancestral/originator events, while those of the IDH family, RTK family and cohesin family are typical secondary events. Establishment of mutational patterns may improve the precision of morphologically-based diagnosis. The comparison between MDS-related diseases (MDS+sAML) and pAML revealed a notably different mutational pattern suggestive of a distinct molecular derivation of these two disease groups. While RTK, IDH family and NPM1 mutations were more frequently observed in the pAML cohort, mutations of SF3B1 and SRSF2, were more common in MDS+sAML. With regard to the connections between individual mutation combinations, RTK mutations were strongly associated with DNMT, but not with RAS family mutations in the pAML cohort, while the mutual association between TET2 and PRC2 family, cohesin family and RUNX1were encountered in the MDS+sAML cohort. Individual mutations may have prognostic significance, including having an impact on survival, either within the entire cohort or within specific subgroups. In the combined MDS cohort, TP53 family mutations were associated with a poor prognosis (HR; 3.65, 95%CI; 1.90-7.01, P 〈 .0001) by univariate analysis. Similar results were found for mutations in TCF4(HR; 7.98, 95%CI; 1.58-10.1, P 〈 .0007). Such an individual approach does not allow for assessment of the impact of less common mutational events. In conclusion, our study continues to indicate the power of NGS in the molecular analysis of MDS. MDS and related disorders show a great deal of pathogenetic molecular overlap, consistent with their morphologic and clinical pictures, but also distinct molecular differences in mutational patterns. Some of the specific mutations are pathognomonic for specific subtypes while some may convey a prognostic rather than discriminatory value. Disclosures: Makishima: Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding. Polprasert:MDS foundation: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.518.518
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 9
    Online Resource
    Online Resource
    Frequent Pathway Mutations of Splicing Machinery in Myelodysplasia
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 458-458
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 458-458
    Abstract: Abstract 458 MDS and related disorders comprise a group of myeloid neoplasms characterized by deregulated blood cell production and a predisposition to AML. Although currently, a number of gene alterations have been implicated in the pathogenesis of MDS, they do not fully explain the pathogenesis of MDS, because many of them are also found in other myeloid malignancies and roughly 20% of MDS cases have no known genetic changes. So, in order to clarify a complete registry of gene mutations in MDS and identify those discriminate MDS from other myeloid neoplasms, we performed whole-exome sequencing of 29 cases showing myelodysplasia. A total of 268 somatic mutations or 9.2 mutations per sample were identified. Among these 41 occurred in recurrent gene targets, which not only included a spectrum of known gene targets in MDS, such as TET2, EZH2, NRAS/KRAS, RUNX1, TP53 and DNMT3A, but also affected previously unknown genes that are commonly mapped to the RNA splicing pathway, including U2AF35, SRSF2 and ZRSR2. Together with additional three (SF3A1, SF3B1 and PRPF40B) found in single cases, 16 (55.2%) of the 29 discovery cases carried a mutation affecting the component of the splicing machinery. To confirm the observation, we examined 9 spliceosome genes for mutations in a large set of myeloid neoplasms (N=582) using a high-throughput mutation screen of pooled DNA followed by confirmation/identification of candidate mutations. In total, 219 mutations were identified in 209 out of the 582 specimens of myeloid neoplasms. Mutations of the splicing machinery were highly specific to diseases showing myelodysplastic features, including 19 of 23 (83%) cases with RARS, 43 of 50 (86%) RCMD-RS, 68 of 155 (44%) other MDS, 48 of 88 (55%) CMML, and 16 of 62 (26%) secondary AML with MDS features with a string preference of SF3B1 mutations to RARS and RCMD-RS and of SRSF2 to CMML, while they were rare in cases with de novo AML (N=151) and MPD (N=53). The mutations among 4 genes, U2AF35 (N = 37), SRSF2 (N = 56), SF3B1 (N = 79) and ZRSR2 (N = 23), explained most of the mutations with a much lower mutational rate for SF3A1 (N = 8), PRPF40B (N = 7), U2AF65 (N = 4) and SF1 (N = 5). Interestingly, mutations in the former three genes showed clear hot spots, indicating a gain-of-function nature of these mutations. On the other hand, two thirds of the ZRSR2 mutations are nonsense or frameshift changes causing premature truncation of the protein. Significantly, these mutations occurred in an almost completely mutually exclusive manner among mutated cases, and commonly affected those components of the splicing complex that are engaged in the 3' splice site recognition during RNA splicing, strongly indicating production of unspliced or aberrantly spliced RNA species are incriminated for the pathogenesis of MDS. In fact, when transduced into HeLa cells, the recurrent S34F U2AF35 mutant induced the increase in the production of unspliced RNA species and elicited the activation of the nonsense mediated decay pathway. Functionally, the U2AF35 mutants seemed to cause deregulated stem cell functions, because CD34(−) KSL cells transduced with various U2AF35 mutants invariably showed reduced chimerism in competitive reconstitution assay. In accordance with this, the S34F U2AF35 mutant lead to suppression of cell growth in a variety of cell types, including HeLa cells, in which expression of the mutant induced a G2/M cell cycle arrest and increased apoptosis. In conclusion, whole-exome sequencing unexpectedly revealed the high frequency of the splicing pathway mutations in MDS and related myeloid neoplasms, providing the first evidence indicating that compromised RNA splicing by gene mutations are responsible for human pathogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.458.458
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
  • 10
    Online Resource
    Online Resource
    Mutational Spectrum Analysis of Interesting Correlation and Interrelationship Between RNA Splicing Pathway and Commonly Targeted Genes in Myelodysplastic Syndrome
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 273-273
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 273-273
    Abstract: Abstract 273 Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms showing a frequent transition to acute myeloid leukemia. Although they are discriminated from de novo AML by the presence of a preleukemic period and dysplastic cell morphology, the difference in molecular genetics between both neoplasms has not been fully elucidated because of the similar spectrum of gene mutations. In this regards, the recent discovery of frequent pathway mutations (45∼90%) involving the RNA splicing machinery in MDS and related myeloid neoplasm with their rare mutation rate in de novo AML provided a novel insight into the distinct molecular pathogenesis of both neoplasms. Thus far, eight components of the RNA splicing machinery have been identified as the targets of gene mutations, among which U2AF35, SF3B1, SRSF2 and ZRSR2 show the highest mutation rates in MDS and CMML. Meanwhile, the frequency of mutations shows a substantial variation among disease subtypes, although the genetic/biological basis for these differences has not been clarified; SF3B1 mutations explain 〉 90% of the spliceosome gene mutations in RARS and RCMD-RS, while mutations of U2AF35 and ZRSR2 are rare in these categories ( 〈 5%) but common in CMML (16%) and MDS without increased ring sideroblasts (20%). On the other hand, SRSF2 mutations are most frequent in CMML (30%), compared with other subtypes ( 〈 10 %) (p 〈 0.001) (Yoshida K, et al, unpublished data). So to obtain an insight into the genetic basis for these difference, we extensively explored spectrums of gene mutations in a set of 161 samples with MDS and related myeloid neoplasms, in which mutations of 10 genes thus far identified as major targets in MDS were examined and their frequencies were compared with regard to the species of mutated components of the splicing machinery. The mutation status of the 161 specimens was determined using the target exon enrichment followed by massively parallel sequencing. In total, 86 mutations were identified in 81(50%) in the 8 components of the splicing machinery. The mutations among 4 genes, U2AF35 (N = 20), SRSF2 (N = 31), SF3B1 (N = 15) and ZRSR2 (N = 10), explained most of the mutations with a much lower mutational rate for SF3A1 (N = 3), PRPF40B (N = 3), U2AF65 (N = 3) and SF1 (N = 1). Conspicuously, higher frequency 4 components of the splicing machinery were mutated in 76 out of the 161 cases (47.2%) in a mutually exclusive manner. On the other hand, 172 mutations of the 10 common targets were identified among 117, including 41 TET2 (25%), 32 RUNX1 (20%), 26 ASXL1 (16%), 24 RAS (NRAS/KRAS) (15%), 22 TP53 (14%), 17 IDH1/2 (10%), 10 CBL (6%) and 10 EZH2 (6%) mutations. We examined the difference between the major spliceosome mutations in terms of the number of the accompanying mutations in the 10 common gene targets. The possible bias from the difference in disease subtypes was compensated by multiple regressions. The SRSF2 mutations are more frequently associated with accompanying gene mutations with a significantly higher number of those mutations (N=29; OR 6.2; 95%CI 1.1–35) compared with that of the U2AF35 mutations (N=14) (p=0.038). Commonly involving the E/A splicing complexes, these splicing pathway mutations lead to compromised 3' splice site recognition. However, individual mutations may still have different impacts on cell functions, which could contribute to the determination of discrete disease phenotypes. It was demonstrated that SRSF2 was involved in the regulation of DNA stability and that depletion of SRSF2 can lead to DNA hypermutability, which may explain the higher number of accompanying gene mutation in SRSF2-mutated cases than cases with other spliceosome gene mutations. In conclusion, it may help to disclosing the genetic basis of MDS and related myeloid neoplasms that highly paralleled resequencing was confirmed SRSF2 mutated case significantly overlapped common mutations. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.273.273
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
Close ⊗
This website uses cookies and the analysis tool Matomo. More information can be found here...