PO.BCS01.15 · 生物信息与计算
利用FLAIR3进行长读长RNA测序的癌症基因变异鉴定与功能解读
Cancer gene variant identification and functional interpretation using long-read RNA sequencing with FLAIR3
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摘要 Abstract
中文摘要
尽管单核苷酸变异（SNV）以及转录和RNA加工的变化通常被分开分析，但综合分析有助于全面理解癌症基因改变如何影响肿瘤发生。在传统的短读长RNA测序中，可变外显子与癌症变异之间的定相会丢失，因为读段长度远短于典型的mRNA转录本（平均>1kb）。在此，我们展示长读长RNA测序（lrRNA-seq）能够鉴定变异所表达于的全长转录本异构体，从而可用于更准确地鉴定致癌变异的功能影响。我们开发了FLAIR3，它利用lrRNA-seq对SNV、基因融合和可变剪接进行整合分析，并预测氨基酸序列的功能变化。我们对三种骨肉瘤细胞系进行了ONT lrRNA-seq，对两例肺腺癌的配对正常和肿瘤组织进行了PacBio lrRNA-seq。随后我们使用FLAIR3鉴定癌症驱动变异，并确定剪接如何调节其表达和功能。在骨肉瘤样本中，FLAIR3揭示了癌症驱动基因中的可变剪接基因融合以及TP53与基因间区的基因融合，预计会导致TP53截短。在肺腺癌中，FLAIR3揭示了致癌性BRAF V600E的异构体偏向性表达。通过对CDKN2A中体细胞SNV的异构体特异性分析，我们发现TP53缺失与CDKN2A p16异构体的错义或无义变异显著共现，但与CDKN2A深度缺失不共现。p16异构体中的破坏性变异在通过TP53发挥功能的p14异构体中不会产生相同的破坏性效应；因此，要完全丧失CDKN2A功能，TP53缺失是必需的。CDKN2A的深度缺失会同时去除p16和p14异构体的功能，因而不需要额外的TP53缺失。这些发现揭示了可变剪接如何与致癌变异的功能相互作用并对其进行调节。
查看英文原文 English abstract
Although the impact of single nucleotide variants (SNVs) and changes in transcription and RNA processing are often analyzed separately, a comprehensive analysis facilitates a complete understanding of how cancer gene alterations impact oncogenesis. In traditional short-read RNA sequencing, phasing of alternative exons and cancer variants is lost because the read lengths are much shorter than typical mRNA transcripts (average > 1kb). Here, we show that long-read RNA-seq (lrRNA-seq) can identify full-length transcript isoforms on which variants are expressed, which can be used to more accurately identify the functional impact of oncogenic variants. We developed FLAIR3, which performs an integrated analysis of SNVs, gene fusions, and alternative splicing using lrRNA-seq and predicts functional changes to the amino acid sequence. We performed ONT lrRNA-seq on three osteosarcoma cell lines and PacBio lrRNA-seq on paired normal and tumor tissue from two lung adenocarcinomas. We then used FLAIR3 to identify cancer driver variants and to determine how splicing modulates their expression and function. In the osteosarcoma samples, FLAIR3 revealed alternatively spliced gene fusions in cancer driver genes and TP53 gene fusions with intergenic regions, predicted to cause TP53 truncations. In the lung adenocarcinomas, FLAIR3 revealed isoform-biased expression of oncogenic BRAF V600E . Through an isoform-specific analysis of somatic SNVs in CDKN2A, we found that TP53 loss significantly co-occurs with CDKN2A missense or nonsense variants of the p16 isoform, but not with CDKN2A deep deletion. Damaging variants in the p16 isoform would not have the same damaging effects in p14 isoform, which functions through TP53; therefore, TP53 loss would be necessary to have complete loss of CDKN2A functions. A deep deletion of CDKN2A removes both p16 and p14 isoform function and would not need to have additional TP53 loss. These findings reveal how alternative splicing interacts with and modulates the function of oncogenic variants.
利益披露 Disclosure
M. Diekhans, None..
C. Vollmers, None..
A. Berger, None..
A. N. Brooks, None.