• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文学位 >Functional validation of recurrent genomic alterations in pediatric acute leukemia.
【24h】

Functional validation of recurrent genomic alterations in pediatric acute leukemia.

机译：小儿急性白血病复发性基因组改变的功能验证。

获取原文
获取原文并翻译 | 示例
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Members of the Downing laboratory previously used a combination of array-based comparative genomic hybridization and targeted gene re-sequencing to identify a number of recurrent mutations in pediatric acute lymphoid leukemia (ALL). The most commonly targeted genes were PAX5, EBF1 , and IKZF. A number of other rarer lesions were also identified, including focal deletions on chromosome 3 that resulted in the loss of two genes, CD200 and BTLA. By mapping the breakpoint of these chromosome 3 deletions in primary ALL blasts and cell lines, I demonstrated that the deletion resulted in the loss of both CD200 and BTLA in all but one case. Moreover, the results of analyzing the sequence at the breakpoints suggested that deletion was the result of aberrant RAG-mediated recombination. Importantly, no point mutations in CD200 or BTLA were detected in any of the analyzed pediatric ALL cases, suggesting that haploinsufficiency of these two genes was likely to contribute to leukemogenesis.;To explore the functional consequence of these deletions, I first analyzed the expression pattern of both genes as a function of normal B-cell development. In agreement with published studies, both genes showed low expression during early B-cell development but were markedly upregulated in mature B cells. To explore whether loss of both genes contributes to leukemogenesis, I crossed mice heterozygous for a Cd200-null allele (Cd200 +/-) with mice heterozygous for a Btla-null allele (Btla +/-) to generate mice that were heterozygous for both genes (Cd200+/-: Btla+/-). I then examined B-cell development in these mice. My analysis revealed no significant alteration in B-cell development in mice heterozygous for either Cd200 or Btla alone. In contrast, the Cd200+/-: Btla+/- mice had a subtle decrease in mature B cells and a modest increase in pre-B cells, suggesting that loss of both genes cooperated to dysregulate normal B-cell development. To determine if this defect could cooperate with other genetic lesions in leukemogenesis, bone marrow cells from wild-type, Cd200 +/-, Btla +/-, and Cd200+/-: Btla+/- mice were transduced with a retrovirus expressing BCR-ABL1 and GFP and then transplanted into lethally irradiated syngeneic mice. As previously published, BCR-ABL1 induces a pre-B cell acute lymphoblastic leukemia in wild-type mice. No significant difference was noted in the penetrance, latency, or disease phenotype of bone marrow cells obtained from mice heterozygous for Cd200 or Btla. In contrast, expression of BCR-ABL1 in marrow obtained from Cd200+/-: Btla+/- mice resulted in a significant decrease in latency and increase in penetrance of ALL without affecting the immunophenotype of the resultant leukemia. These data suggest that the co-deletion of CD200 and BTLA induces a subtle block in normal B-cell development and can cooperate with the expression of BCR-ABL1 in leukemogenesis.;In a second series of experiments, I focused on exploring the role of a focal recurrent amplification of chromosome 8q24.21 in AML that was initially identified in the Downing laboratory. The minimally altered region contains a single putative gene called CCDC26 that was initially identified as the target of retroviral integration in a forward genetic screen to identify genes whose alteration blocked retinoic acid-induced myeloid cell differentiation. This region is amplified in approximately 4% of AMLs and is also affected by focal amplifications or deletions in rare cases of pediatric ALL. To further characterize this deletion, I used a variety of approaches, including tiling array CGH, whole-genome sequencing, and transcriptome sequencing of mRNA and nuclear and cytoplasmic large-intervening non-coding RNAs (Linc-RNAs), to determine the target of these leukemia-associated copy-number alterations (CNAs). Our analysis revealed the expression of altered splice variants of CCDC26 in leukemia cells that lacked an open reading frame and showed no change in expression as a result of CNAs. These data suggest that CCDC26 is not the primary target of the CNAs. Our transcriptome analysis, coupled with detailed bioinformatic analysis of public databases of chromatin modification in leukemic cells, identified a 1.2-kb unspliced nuclear Linc-RNA whose expression was increased in leukemia cell lines and primary AML samples that contained a focal 8q24.21 amplification. Knockdown of this Linc-RNA with two independent antisense oligonucleotides resulted in a subtle decrease in cell viability. The complexity of the Linc-RNAs encoded by the genomic region, coupled with the lack of homology between mice and humans in this genomic region, makes it very challenging to define the role of this region in leukemogenesis. (Abstract shortened by UMI.)
机译：唐宁实验室的成员以前使用基于阵列的比较基因组杂交和靶向基因重测序的组合来鉴定小儿急性淋巴白血病（ALL）中的许多复发突变。最常见的靶向基因是PAX5，EBF1和IKZF。还鉴定出许多其他罕见病灶，包括3号染色体上的局部缺失，导致两个基因CD200和BTLA丢失。通过绘制原代ALL细胞和细胞系中这些3号染色体缺失的断点，我证明了该缺失导致除一种情况外，所有其他基因都导致了CD200和BTLA的丢失。此外，在断点处分析序列的结果表明缺失是异常RAG介导的重组的结果。重要的是，在所有已分析的儿科ALL病例中均未检测到CD200或BTLA的点突变，这表明这两个基因的单倍体不足可能促进了白血病的发生。为了探讨这些缺失的功能后果，我首先分析了表达模式两个基因的表达与正常B细胞​​发育的关系。与已发表的研究一致，这两个基因在早期B细胞发育过程中均显示低表达，但在成熟B细胞中明显上调。为了探究这两个基因的缺失是否有助于白血病的发生，我将Cd200-null等位基因（Cd200 +/-）杂合的小鼠与Btla-null等位基因（Btla +/-）杂合的小鼠杂交，以产生对这两个杂合的小鼠基因（Cd200 +/-：Btla +/-）。然后，我检查了这些小鼠的B细胞发育。我的分析显示，对于单独的Cd200或Btla，杂合的小鼠B细胞发育没有显着改变。相比之下，Cd200 +/-：Btla +/-小鼠的成熟B细胞略有减少，而前B细胞则有适度的增加，这表明这两个基因的丧失协同调节了正常B细胞​​的发育。为了确定该缺陷是否可以与白血病发生中的其他遗传损伤协同作用，用表达BCR-ABL1的逆转录病毒转导野生型，Cd200 +/-，Btla +/-和Cd200 +/-的骨髓细胞：和GFP，然后移植到经致命照射的同系小鼠中。如先前公开的那样，BCR-ABL1在野生型小鼠中诱发B前细胞急性淋巴细胞白血病。从Cd200或Btla杂合的小鼠获得的骨髓细胞的外在性，潜伏期或疾病表型方面没有发现显着差异。相反，从Cd200 +/-：Btla +/-小鼠获得的骨髓中BCR-ABL1的表达导致ALL潜伏期显着减少和渗透率增加，而不会影响所得白血病的免疫表型。这些数据表明CD200和BTLA的共同缺失在正常B细胞​​发育中诱导了微小的阻滞，并且可以与BCR-ABL1的表达在白血病发生中协同作用。在唐宁实验室最初发现的AML中8q24.21染色体的局部复发性扩增。最小变化的区域包含一个称为CCDC26的推定基因，该基因最初被确定为正向遗传筛选中逆转录病毒整合的靶标，以鉴定其改变会阻止视黄酸诱导的髓样细胞分化的基因。该区域在大约4％的AML中被扩增，并且在罕见的儿科ALL病例中还受到病灶扩增或缺失的影响。为了进一步表征这种缺失，我使用了多种方法，包括平铺阵列CGH，全基因组测序以及mRNA和核及细胞质大分子非编码RNA（Linc-RNA）的转录组测序，以确定靶点。这些与白血病相关的拷贝数改变（CNA）。我们的分析表明，缺乏开放阅读框的白血病细胞中CCDC26剪接变体的表达发生变化，并且由于CNA的影响，表达没有变化。这些数据表明，CCDC26不是CNA的主要目标。我们的转录组分析，以及对白血病细胞染色质修饰公共数据库的详细生物信息学分析，确定了1.2kb未剪接的核Linc-RNA，其表达在白血病细胞系和包含局灶性8q24.21扩增的原代AML样品中表达增加。用两个独立的反义寡核苷酸敲低该Linc-RNA导致细胞活力的轻微降低。基因组区域编码的Linc-RNA的复杂性，再加上该基因组区域中的小鼠与人类之间缺乏同源性，因此定义该区域在白血病发生中的作用非常具有挑战性。 （摘要由UMI缩短。）

著录项

  • 作者

    Cheng, Jinjun.;

  • 作者单位

    The University of Tennessee Health Science Center.;

  • 授予单位 The University of Tennessee Health Science Center.;
  • 学科 Biology Molecular.;Health Sciences Oncology.;Biology Genetics.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 99 p.
  • 总页数 99
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号