Long-read DNA and cDNA sequencing identify cancer-predisposing deep intronic variation in tumor-suppressor genes

IF 6.2 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Genome research Pub Date : 2024-09-13 DOI:10.1101/gr.279158.124
Suleyman Gulsuner, Amal AbuRayyan, Jessica B. Mandell, Ming K. Lee, Greta V. Bernier, Barbara M. Norquist, Sarah B. Pierce, Mary-Claire King, Tom Walsh
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Abstract

The vast majority of deeply intronic genomic variants are benign, but some extremely rare or private deep intronic variants lead to exonification of intronic sequence with abnormal transcriptional consequences. Damaging variants of this class are likely underreported as causes of disease for several reasons: Most clinical DNA and RNA testing does not include full intronic sequences; many of these variants lie in complex repetitive regions that cannot be aligned from short-read whole-genome sequence; and, until recently, consequences of deep intronic variants were not accurately predicted by in silico tools. We evaluated the frequency and consequences of rare deep intronic variants for families severely affected with breast, ovarian, pancreatic, and/or metastatic prostate cancer, but with no causal variant identified by any previous genomic or cDNA-based approach. For 10 tumor-suppressor genes, we used multiplexed adaptive sampling long-read DNA sequencing and cDNA sequencing, based on patient-derived DNA and RNA, to systematically evaluate deep intronic variation. We identified all variants across the full genomic loci of targeted genes, applied the in silico tools SpliceAI and Pangolin to predict variants of functional consequence, and then carried out long-read cDNA sequencing to identify aberrant transcripts. For eight of the 120 (6%) previously unsolved families, rare deep intronic variants in BRCA1, PALB2, and ATM create intronic pseudoexons that are spliced into transcripts, leading to premature truncations. These results suggest that long-read DNA and cDNA sequencing can be integrated into variant discovery, with strategies for accurately characterizing pathogenic variants.
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长读DNA和cDNA测序确定肿瘤抑制基因中易致癌的深层内含子变异
绝大多数深度内含子基因组变异都是良性的,但一些极其罕见或私密的深度内含子变异会导致内含子序列的外显子化,从而产生异常的转录后果。由于多种原因,这类损伤性变异很可能未被充分报告为致病原因:大多数临床 DNA 和 RNA 检测并不包括完整的内含子序列;许多此类变异位于复杂的重复区域,无法通过短读数全基因组序列进行比对;直到最近,深度内含子变异的后果也无法通过硅学工具准确预测。我们评估了受乳腺癌、卵巢癌、胰腺癌和/或转移性前列腺癌严重影响的家族中罕见深部内含子变异的频率和后果,但以前的任何基因组学或基于 cDNA 的方法都没有发现因果变异。对于 10 个肿瘤抑制基因,我们使用了基于患者 DNA 和 RNA 的多重自适应采样长读数 DNA 测序和 cDNA 测序来系统评估深层内含子变异。我们确定了目标基因全基因组位点上的所有变异,应用 Silico 工具 SpliceAI 和 Pangolin 预测功能性变异,然后进行长读程 cDNA 测序以确定异常转录本。在 120 个先前未解决的家族中,有 8 个家族(6%)的 BRCA1、PALB2 和 ATM 中的罕见深内含子变异产生了内含子假外显子,这些假外显子被剪接到转录本中,导致过早截断。这些结果表明,长线程DNA和cDNA测序可被整合到变异发现中,其策略可准确表征致病变异。
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来源期刊
Genome research
Genome research 生物-生化与分子生物学
CiteScore
12.40
自引率
1.40%
发文量
140
审稿时长
6 months
期刊介绍: Launched in 1995, Genome Research is an international, continuously published, peer-reviewed journal that focuses on research that provides novel insights into the genome biology of all organisms, including advances in genomic medicine. Among the topics considered by the journal are genome structure and function, comparative genomics, molecular evolution, genome-scale quantitative and population genetics, proteomics, epigenomics, and systems biology. The journal also features exciting gene discoveries and reports of cutting-edge computational biology and high-throughput methodologies. New data in these areas are published as research papers, or methods and resource reports that provide novel information on technologies or tools that will be of interest to a broad readership. Complete data sets are presented electronically on the journal''s web site where appropriate. The journal also provides Reviews, Perspectives, and Insight/Outlook articles, which present commentary on the latest advances published both here and elsewhere, placing such progress in its broader biological context.
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