公开(公告)号：US20140227705A1

公开(公告)日：2014-08-14

申请号：US14111715

申请日：2012-04-12

申请人： Bert Vogelstein ,  Kenneth W. Kinzler ,  Nickolas Papadopoulos ,  Isaac Kinde

发明人： Bert Vogelstein ,  Kenneth W. Kinzler ,  Nickolas Papadopoulos ,  Isaac Kinde

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6874 ,  C12Q1/6806 ,  C12Q1/6869 ,  C12Q1/6876 ,  C12Q2563/179 ,  C12Q2600/158 ,  C12Q2535/122 ,  C12Q2565/514

摘要： The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≧95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

摘要翻译： 存在于DNA模板的一小部分中的突变的鉴定对于在几个生物医学研究领域的进展是必不可少的。 尽管大规模并行测序仪器原则上非常适合于此任务，但是这些仪器中的错误率通常太高，无法确定罕见的变体。 我们在这里描述了一种可以显着增加大规模并行测序仪器对此目的的灵敏度的方法。 这种方法的一个例子是（Safe-Sequencing System），称为“Safe-SeqS”，包括（i）将唯一标识符（UID）分配给每个模板分子; （ii）每个唯一标记的模板分子的扩增以产生UID家族; 和（iii）扩增产物的冗余测序。 具有相同UID的PCR片段是真正的突变体（“超突变体”），如果其≥95％含有相同的突变。 我们说明了这种方法用于确定聚合酶的保真度，体外合成的寡核苷酸的准确性以及正常细胞的核和线粒体基因组中突变的流行率的效用。

公开(公告)号：US20140227271A1

公开(公告)日：2014-08-14

申请号：US14129850

申请日：2012-06-28

申请人： Hai Yan ,  Darell Bigner ,  Bert Vogelstein ,  Kenneth W. Kinzler ,  Alan Meeker ,  Ralph Hruban ,  Nickolas Papadopoulos ,  Luis Diaz ,  Yuchen Jiao

发明人： Hai Yan ,  Darell Bigner ,  Bert Vogelstein ,  Kenneth W. Kinzler ,  Alan Meeker ,  Ralph Hruban ,  Nickolas Papadopoulos ,  Luis Diaz ,  Yuchen Jiao

IPC分类号： C12Q1/68 ,  C07K16/40 ,  C12N15/113 ,  G01N33/574

CPC分类号： C12Q1/6886 ,  C07K16/40 ,  C12N15/1137 ,  C12Q2600/118 ,  C12Q2600/156 ,  G01N33/57407

摘要： We determined the sequence of ATRX and DAXX in 447 cancers from various sites. We found mutations most commonly in pediatric glioblastoma multiformae (GBM) (11.1%), adult GBM (6.5%), oligodendrogliomas (7.7%) and medulloblastomas (1.5%); and showed that Alternative Lengthening of Telomeres (ALT), a telomerase-independent telomere maintenance mechanism found in cancers that have not activated telomerase, perfectly correlated with somatic mutations of either gene. In contrast, neuroblastomas, and adenocarcinomas of the ovary, breast, and pancreas were negative for mutations in ATRX and DAXX. Alterations in ATRX or DAXX define a specific molecular pathway that is closely associated with an alternative telomere maintenance function in human cancers.

摘要翻译： 我们确定了来自各个位点的447例癌症中ATRX和DAXX的序列。 我们发现多形性小儿多形性成胶质细胞瘤（GBM）（11.1％），成人GBM（6.5％），少突胶质细胞瘤（7.7％）和成神经管细胞瘤（1.5％）中最常见的突变; 并且表明，在未激活端粒酶的癌症中发现端粒酶不依赖端粒维持机制的替代延长端粒（ALT）与任一基因的体细胞突变完全相关。 相比之下，成骨细胞瘤和卵巢，乳腺和胰腺腺癌对于ATRX和DAXX中的突变是阴性的。 ATRX或DAXX中的改变定义了与人类癌症中替代端粒维持功能密切相关的特定分子途径。

公开(公告)号：US08110356B2

公开(公告)日：2012-02-07

申请号：US12728958

申请日：2010-03-22

申请人： Nicholas C. Nicolaides ,  Luigi Grasso ,  Phillip M. Sass ,  Kenneth W. Kinzler ,  Bert Vogelstein

发明人： Nicholas C. Nicolaides ,  Luigi Grasso ,  Phillip M. Sass ,  Kenneth W. Kinzler ,  Bert Vogelstein

IPC分类号： C12Q1/68 ,  C12N15/82

CPC分类号： C12N15/1024 ,  C07K14/415 ,  C12N15/8216 ,  C12N15/8241

摘要： Blockade of mismatch repair in a plant can lead to hypermutation and a new genotype and/or phenotype. One approach used to generate hypermutable plants is through the expression of dominant negative alleles of mismatch repair genes in transgenic plants or derived cells. By introducing these genes into cells and transgenic plants, new cell lines and plant varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. Moreover, methods to inhibit the expression and activity of endogenous plant MMR genes and their encoded products are also useful to generate hypermutable plants.

摘要翻译： 植物中错配修复的阻断可导致超突变和新的基因型和/或表型。 用于产生超可变植物的一种方法是通过在转基因植物或衍生细胞中表达错配修复基因的显性负等位基因。 通过将这些基因引入细胞和转基因植物，可以比通过依赖于突变的自然速率更有效地制备具有新颖和有用性质的新细胞系和植物品种。 此外，抑制内源植物MMR基因及其编码产物的表达和活性的方法也可用于产生高可变性植物。

公开(公告)号：US20120009573A1

公开(公告)日：2012-01-12

申请号：US13131413

申请日：2009-12-02

申请人： Bert Vogelstein ,  Kenneth W. Kinzler ,  Yiping He ,  Victor Velculescu ,  Nickolas Papadopoulos

发明人： Bert Vogelstein ,  Kenneth W. Kinzler ,  Yiping He ,  Victor Velculescu ,  Nickolas Papadopoulos

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6844 ,  G06F19/22 ,  C12Q2539/113 ,  C12Q2523/125 ,  C12Q2521/107

摘要： Transcription in mammalian cells can be assessed at a genome-wide level, but it has been difficult to reliably determine whether individual transcripts are derived from the Plus- or Minus-strands of chromosomes. This distinction can be critical for understanding the relationship between known transcripts (sense) and the complementary antisense transcripts that may regulate them. Here we describe a technique that can be used to (i) identify the DNA strand of origin for any particular RNA transcript and (ii) quantify the number of sense and antisense transcripts from expressed genes at a global level. We examined five different human cell types and in each case found evidence for antisense transcripts in 2900 to 6400 human genes. The distribution of antisense transcripts was distinct from that of sense transcripts, was non-random across the genome, and differed among cell types. Anti-sense transcripts thus appear to be a pervasive feature of human cells, suggesting that they are a fundamental component of gene regulation.

摘要翻译： 哺乳动物细胞中的转录可以在全基因组范围内进行评估，但是难以可靠地确定个体转录本是否衍生自染色体的加号或阴影链。 这种区别对于了解已知转录物（有义）和可能调节它们的互补反义转录物之间的关系可能是至关重要的。 在这里，我们描述了一种可用于（i）识别任何特定RNA转录物的DNA链起源的技术，（ii）在全球范围内量化来自表达基因的正义和反义转录本的数量。 我们检查了五种不同的人类细胞类型，并且在每种情况下都发现2900至6400个人类基因中的反义转录物的证据。 反义转录物的分布与有义转录物的分布不同，在基因组中是非随机的，并且在细胞类型之间不同。 因此，反义转录物似乎是人类细胞的普遍特征，表明它们是基因调控的基本组成部分。

公开(公告)号：US08039210B2

公开(公告)日：2011-10-18

申请号：US11596349

申请日：2005-05-16

申请人： Zhenghe Wang ,  Victor Velculescu ,  Kenneth W. Kinzler ,  Bert Vogelstein

发明人： Zhenghe Wang ,  Victor Velculescu ,  Kenneth W. Kinzler ,  Bert Vogelstein

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6886 ,  C12N9/16 ,  C12Q2600/112 ,  C12Q2600/136 ,  C12Q2600/156 ,  C12Q2600/158 ,  C12Y301/03048 ,  G01N33/5011 ,  G01N2333/916

摘要： Tyrosine phosphorylation, regulated by protein tyrosine phosphatases (PTPs) and kinases (PTKs), is important in signaling pathways underlying tumorigenesis. A mutational analysis of the tyrosine phosphatase gene superfamily in human cancers identified 83 somatic mutations in six PTPs (PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, PTPN14) affecting 26% of colorectal cancers and a smaller fraction of lung, breast and gastric cancers. Fifteen mutations were nonsense, frameshift or splice site alterations predicted to result in truncated proteins lacking phosphatase activity. Five missense mutations in the most commonly altered PTP (PTPRP) were biochemically examined and found to reduce phosphatase activity. Expression of wild-type but not a mutant PTPRT in human cancer cells inhibited cell growth. These observations suggest that the tyrosine phosphatase genes are tumor suppressor genes, regulating cellular pathways that may be amenable to therapeutic intervention.

摘要翻译： 由蛋白酪氨酸磷酸酶（PTP）和激酶（PTK）调节的酪氨酸磷酸化在肿瘤发生的信号通路中是重要的。 人类癌症中酪氨酸磷酸酶基因超家族的突变分析鉴定了影响26％结肠直肠癌和较小部分肺癌，乳腺癌和胃癌的6种PTPs（PTPRF，PTPRG，PTPRT，PTPN3，PTPN13，PTPN14）中的83个体细胞突变。 十五个突变是无义，移位或剪接位点改变，预计会导致截短的蛋白质缺乏磷酸酶活性。 在生物化学检查中发现最常改变的PTP（PTPRP）中的五个错义突变被发现可以降低磷酸酶活性。 野生型但不是突变PTPRT在人类癌细胞中的表达抑制细胞生长。 这些观察结果表明酪氨酸磷酸酶基因是肿瘤抑制基因，调节可能适合于治疗干预的细胞途径。

公开(公告)号：US08029764B2

公开(公告)日：2011-10-04

申请号：US10487934

申请日：2002-09-09

申请人： Phillip Buckhaults ,  Kenneth W. Kinzler ,  Bert Vogelstein

发明人： Phillip Buckhaults ,  Kenneth W. Kinzler ,  Bert Vogelstein

IPC分类号： A61K49/00 ,  C12Q1/37

CPC分类号： C07K14/495 ,  C07K14/47 ,  C07K14/705

摘要： Particular genes are aberrantly and consistently expressed in both adenomas and carcinomas of the colon. Products of such genes provide stool and serum markers for colorectal neoplasia. One particular tumor marker, Renal Dipeptidase (RDP), is expressed at high levels in tumors and at greatly reduced levels in normal tissues. The elevated expression of RDP occurs early and remains elevated during the neoplastic process. RDP may therefore be especially useful as a diagnostic tool for the early detection of colorectal neoplasia, even of presymptomatic colorectal neoplasia.

摘要翻译： 特定的基因在结肠的腺瘤和癌中异常一致地表达。 这些基因的产物提供结肠直肠肿瘤的粪便和血清标志物。 一个特定的肿瘤标志物肾二肽酶（RDP）在肿瘤中以高水平表达，并在正常组织中以大大降低的水平表达。 RDP升高的表达早期发生，并在肿瘤过程中保持升高。 因此，RDP可以作为用于早期检测结肠直肠肿瘤，甚至是症状性结直肠肿瘤的诊断工具。

公开(公告)号：US20110059434A1

公开(公告)日：2011-03-10

申请号：US11920860

申请日：2006-05-23

申请人： Donald William Parsons ,  Tian-li Wang ,  Yardena Samuels ,  Alberto Bardelli ,  Christopher Lengauer ,  Victor Velculescu ,  Kenneth W. Kinzler ,  Bert Vogelstein

发明人： Donald William Parsons ,  Tian-li Wang ,  Yardena Samuels ,  Alberto Bardelli ,  Christopher Lengauer ,  Victor Velculescu ,  Kenneth W. Kinzler ,  Bert Vogelstein

IPC分类号： C12Q1/68 ,  C12Q1/48 ,  C12N9/12 ,  G01N33/574

CPC分类号： C12Q1/6883 ,  C12Q1/485 ,  C12Q2600/136 ,  C12Q2600/156 ,  G01N33/5044 ,  G01N33/57419

摘要： Given the important role of protein kinases in pathways affecting cellular growth and invasion, we have analyzed 340 serine/threonine kinases for genetic mutations in colorectal cancers. Mutations in eight genes were identified, including three members of the phosphatidylinositol-3-kinase (PI3K) pathway; the alterations in the latter genes each occurred in different tumors and did not overlap with mutations in PIK3CA or other non-serine-threonine kinase (STK) members of the PI3K pathway, suggesting that mutations in any of these genes had equivalent tumorigenic effects. These data demonstrate that the PI3K pathway is a major target for mutational activation in colorectal cancers and provide new opportunities for therapeutic intervention.

摘要翻译： 鉴于蛋白激酶在影响细胞生长和侵袭的途径中的重要作用，我们已经分析了340个丝氨酸/苏氨酸激酶在结肠直肠癌中的遗传突变。 鉴定出8个基因的突变，包括磷脂酰肌醇-3-激酶（PI3K）通路的3个成员; 后一种基因的改变各自发生在不同的肿瘤中，并且不与PIK3CA或PI3K途径的其他非丝氨酸 - 苏氨酸激酶（STK）成员的突变重叠，这表明任何这些基因中的突变具有相等的致瘤作用。 这些数据表明，PI3K途径是结肠直肠癌突变激活的主要靶标，为治疗干预提供新的机会。

公开(公告)号：US20100316995A1

公开(公告)日：2010-12-16

申请号：US12377073

申请日：2007-08-13

申请人： Tobias Sjoblom ,  Sian Jones ,  D. Williams Parsons ,  Laura D. Wood ,  Jimmy Lin ,  Thomas Barber ,  Diana Mandelker ,  Bert Vogelstein ,  Kenneth W. Kinzler ,  Victor E. Velculesu

发明人： Tobias Sjoblom ,  Sian Jones ,  D. Williams Parsons ,  Laura D. Wood ,  Jimmy Lin ,  Thomas Barber ,  Diana Mandelker ,  Bert Vogelstein ,  Kenneth W. Kinzler ,  Victor E. Velculesu

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6886 ,  C12Q2600/106 ,  C12Q2600/156

摘要： Analysis of 13,023 genes in 11 breast and 11 colorectal cancers revealed that individual tumors accumulate an average of ˜90 mutant genes but that only a subset of these contribute to the neoplastic process. Using stringent criteria to delineate this subset, we identified 189 genes (average of 11 per tumor) that were mutated at significant frequency. The vast majority of these genes were not known to be genetically altered in tumors and are predicted to affect a wide range of cellular functions, including transcription, adhesion, and invasion. These data define the genetic landscape of two human cancer types, provide new targets for diagnostic and therapeutic intervention and monitoring.

摘要翻译： 分析11个乳房和11个结直肠癌中的13,023个基因显示，个体肿瘤平均累积〜90个突变基因，但只有其中一个子集对肿瘤过程有贡献。 使用严格的标准来描绘这一亚型，我们确定了189个基因（平均每个肿瘤11个），以显着的频率突变。 这些基因绝大多数不知道在肿瘤中被遗传改变，并被预测会影响广泛的细胞功能，包括转录，粘附和侵袭。 这些数据定义了两种人类癌症类型的遗传景观，为诊断和治疗干预和监测提供了新的目标。

公开(公告)号：US07759121B2

公开(公告)日：2010-07-20

申请号：US11930400

申请日：2007-10-31

申请人： Nicholas C. Nicolaides ,  Philip M. Sass ,  Luigi Grasso ,  Bert Vogelstein ,  Kenneth W. Kinzler

发明人： Nicholas C. Nicolaides ,  Philip M. Sass ,  Luigi Grasso ,  Bert Vogelstein ,  Kenneth W. Kinzler

IPC分类号： C12N15/01

CPC分类号： C12N15/81

摘要： Yeast cells are mutagenized to obtain desirable mutants. Mutagenesis is mediated by a defective mismatch repair system which can be enhanced using conventional exogenously applied mutagens. Yeast cells with the defective mismatch repair system are hypermutable, but after selection of desired mutant yeast strains, they can be rendered genetically stable by restoring the mismatch repair system to proper functionality.

摘要翻译： 酵母细胞被诱变以获得所需的突变体。 诱变由缺陷错配修复系统介导，可以使用常规的外源应用诱变剂进行增强。 具有缺陷错配修复系统的酵母细胞是可超过可变的，但是在选择所需的突变酵母菌株之后，可以通过将错配修复系统恢复到适当的功能来使它们变得遗传稳定。

公开(公告)号：US07704689B2

公开(公告)日：2010-04-27

申请号：US11128420

申请日：2005-05-13

申请人： Nicholas C. Nicolaides ,  Luigi Grasso ,  Philip M. Sass ,  Kenneth W. Kinzler ,  Bert Vogelstein

发明人： Nicholas C. Nicolaides ,  Luigi Grasso ,  Philip M. Sass ,  Kenneth W. Kinzler ,  Bert Vogelstein

IPC分类号： C12Q1/68

CPC分类号： C12N15/1024 ,  C07K14/415 ,  C12N15/8216 ,  C12N15/8241

摘要： Blockade of mismatch repair in a plant can lead to hypermutation and a new genotype and/or phenotype. One approach used to generate hypermutable plants is through the expression of dominant negative alleles of mismatch repair genes in transgenic plants or derived cells. By introducing these genes into cells and transgenic plants, new cell lines and plant varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. Moreover, methods to inhibit the expression and activity of endogenous plant MMR genes and their encoded products are also useful to generate hypermutable plants.

摘要翻译： 植物中错配修复的阻断可导致超突变和新的基因型和/或表型。 用于产生超可变植物的一种方法是通过在转基因植物或衍生细胞中表达错配修复基因的显性负等位基因。 通过将这些基因引入细胞和转基因植物，可以比通过依赖于突变的自然速率更有效地制备具有新颖和有用性质的新细胞系和植物品种。 此外，抑制内源植物MMR基因及其编码产物的表达和活性的方法也可用于产生高可变性植物。