LOS ANGELES – At the annual meeting of the American College of Medical Genetics and Genomics held last week, researchers showed how the simultaneous detection of somatic mutations and alternate splice variants could be used to identify mechanisms of cancer drug resistance and may facilitate more personalized cancer therapy in the future.

洛杉矶——在上周举行的美国医学遗传学和基因组学学院年度会议上，研究人员展示了同时检测体细胞突变和选择性剪接变异如何能够用于识别癌症药物耐药机制，并可能在未来促进更加个性化的癌症治疗。

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Colette Felton, a postdoctoral researcher at the University of California, Santa Cruz, presented data on FLAIR3, a computational tool used to detect somatic variants, gene fusions, and gene fusion isoforms from long-read RNA-seq data.

加州大学圣克鲁兹分校的博士后研究员科莱特·费尔顿展示了关于FLAIR3的数据，FLAIR3是一种用于从长读段RNA-seq数据中检测体细胞变异、基因融合和基因融合异构体的计算工具。

Felton said in her talk that one of the drawbacks to short-read RNA-seq is that it is not the optimal technology for getting a full picture of a person's mutational landscape. She likened its use in detecting cancer gene alterations to a Buddhist parable of many blind men trying to describe an elephant.

费尔顿在她的演讲中表示，短读长 RNA-seq 的一个缺点是它并不是获取一个人突变全景的最佳技术。她将它用于检测癌症基因改变比作一个佛教寓言，讲述了许多盲人试图描述一头大象的故事。

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'If everyone is touching a different part of the elephant,' she said, 'then everyone thinks they're seeing a different thing.' Long-read sequencing, she continued, facilitates a more complete picture, enabling researchers 'to see the whole elephant.'

“如果每个人都触摸大象的不同部分，”她说，“那么每个人都会认为他们看到的是不同的东西。” 她继续说道，长读长测序有助于呈现更完整的图景，使研究人员“能够看到整个大象”。

Long-read sequencing has attracted considerable commercial and research interest. The Human Genome Structural Variation Consortium, for example,

长读长测序已经吸引了大量的商业和研究兴趣。例如，人类基因组结构变异联盟（The Human Genome Structural Variation Consortium），

recently combined

最近合并

both short- and long-read sequencing information to release a variant database, and late last year,

结合短读和长读测序信息来发布一个变异数据库，并且在去年年底，

Pacific Biosciences released

Pacific Biosciences 发布了

a lower-throughput benchtop HiFi sequencer.

一款低通量的台式HiFi测序仪。

The Brooks Lab at UCSC, where Felton works, has been developing computational tools for analyzing long-read sequencing data for some years now. The lab's first FLAIR algorithm was designed to narrow RNA-sequencing reads down to a few high-confidence isoforms, and

加州大学圣克鲁兹分校的布鲁克斯实验室，费尔顿所在的实验室，多年来一直在开发用于分析长读长测序数据的计算工具。该实验室的第一个FLAIR算法旨在将RNA测序读数缩小到几个高可信度的同工型。

FLAIR2

FLAIR2

further improved splice detection by incorporating RNA-editing data.

通过整合RNA编辑数据，进一步改进了剪接检测。

Felton said that one key aspect of FLAIR3 was that by combining single-nucleotide variants with gene fusions and their isoforms, the algorithm returns a 'more nuanced functional interpretation of cancer variants.' She demonstrated FLAIR3's capabilities with examples from studies done in osteosarcoma and lung cancer patients.

费尔顿说，FLAIR3 的一个关键方面是，通过将单核苷酸变异与基因融合及其异构体结合，该算法能够“对癌症变异进行更细致的功能解读”。她通过骨肉瘤和肺癌患者研究中的例子展示了 FLAIR3 的功能。

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Osteosarcoma provided a natural application of FLAIR3, Felton said, as it is a cancer primarily driven by structural variants.

费尔顿说，骨肉瘤为FLAIR3提供了一个天然的应用场景，因为这是一种主要由结构变异驱动的癌症。

Felton and her colleagues investigated three osteosarcoma patients who had P10 loss, CCNE1 amplification, and MYC and CDK4 amplifications, respectively, and were sequenced via Pacific Bioscience's HiFi long-read RNA-sequencing platform.

费尔顿和她的同事调查了三名骨肉瘤患者，他们分别出现了P10缺失、CCNE1扩增以及MYC和CDK4扩增，并通过太平洋生物科学公司的HiFi长读RNA测序平台进行了测序。

'The question we are asking with this study is, what new alterations in druggable cancer genes can we identify through long-read RNA sequencing,' she said.

“我们通过这项研究提出的问题是，我们能通过长读长RNA测序识别出哪些新的可成药癌症基因的改变，”她说道。

Felton showed that the patient with the CCNE1 amplification was actually found to have a fusion in that gene's three prime untranslated region. While this doesn't affect the gene's protein-coding region, Felton said that it enables them to see that only one haplotype is being expanded in the pericentromeric region, which provides a more detailed understanding of its mechanism of action.

费尔顿展示了那个CCNE1基因扩增的患者实际上被发现该基因的三prime非翻译区存在融合。虽然这不会影响基因的蛋白质编码区，但费尔顿表示，这使他们能够看到只有一个单倍型在近中心体区域被扩展，从而提供了对其作用机制更详细的了解。

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In the MYC amplification, the team identified a splicing change leading to an altered protein product. This same patient also showed a 24 base pair deletion in the KEAP1 gene that led to a 10 amino acid deletion in the KEAP1 protein.

在MYC扩增中，研究团队发现了一个导致蛋白质产物改变的剪接变化。同一患者还在KEAP1基因中出现了一个24个碱基对的缺失，这导致了KEAP1蛋白中10个氨基酸的缺失。

'The really crazy thing about this,' Felton said, 'is that this is completely undetectable by short-read sequencing. A deletion of this size actually causes short reads to fail to align to the locus, while long reads align around it and allow you to detect this variant.'

“这件事真正疯狂的地方在于，”费尔顿说，“这是完全无法通过短读长测序检测到的。这种大小的缺失实际上会导致短读长无法与该位点对齐，而长读长则可以绕过它并对齐，使你能够检测到这个变异。”

In collaboration with researchers at the Fred Hutchinson Cancer Research Center, Felton and her colleagues are also currently studying alterations in lung cancer patients. She presented findings from one such patient who had altered splicing in the BRAF gene, causing increased usage of one particular isoform of that gene called BRAF-220.

在与弗雷德·哈钦森癌症研究中心的研究人员合作下，费尔顿和她的同事目前也在研究肺癌患者的基因变化。她展示了一位这样的患者的发现，该患者在BRAF基因上存在剪接变异，导致该基因一种特定的异构体BRAF-220的使用增加。

The investigators discovered that a cancer-driving BRAF variant was only expressed on the BRAF-220 isoform..

研究人员发现，一种驱动癌症的 BRAF 变体仅在 BRAF-220 同工型上表达。

The ability to identify allele-specific expression, Felton said, should facilitate treatment.

费尔顿说，识别等位基因特异性表达的能力应该有助于治疗。

'BRAF-220 is the canonical isoform for BRAF,' she said. 'So in this case we can be relatively confident in recommending a BRAF inhibitor as a treatment and not worrying about there being inherent resistance through the alternative splicing of this gene.'

“BRAF-220是BRAF的典型同工型，”她说。“因此在这种情况下，我们可以相对有信心地推荐使用BRAF抑制剂作为治疗方法，而不必担心由于该基因的可变剪接而产生固有的耐药性。”

Felton is currently preparing a manuscript and told

费尔顿目前正在准备一份手稿并表示

GenomeWeb

基因组网络

that she is now planning a study of lung cancer variants in a larger cohort of roughly 60 patients. Although it is too early to make concrete plans, she added that her lab does hope to carry these studies forward to the clinical stage.

她现在正计划在更大约60名患者群体中研究肺癌变异。尽管现在做出具体计划为时尚早，她补充说她的实验室确实希望将这些研究推进到临床阶段。