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.

中文翻译：

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长读长 DNA 和 cDNA 测序可识别肿瘤抑制基因中易患癌症的深内含子变异


绝大多数深度内含子基因组变异是良性的，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。由于以下几个原因，这类破坏性变异可能被低估为疾病原因：大多数临床 DNA 和 RNA 检测不包括完整的内含子序列;其中许多变异位于复杂的重复区域，无法与短读长全基因组序列进行比对;而且，直到最近，计算机工具还无法准确预测深度内含子变体的后果。我们评估了罕见的深内含子变异的频率和后果，这些变异对严重受乳腺癌、卵巢癌、胰腺癌和/或转移性前列腺癌影响的家庭，但没有通过任何先前的基因组或基于 cDNA 的方法确定因果变异。对于 10 个肿瘤抑制基因，我们使用基于患者来源的 DNA 和 RNA 的多重自适应采样长读长 DNA 测序和 cDNA 测序来系统评价深度内含子变异。我们鉴定了目标基因完整基因组位点中的所有变异，应用计算机工具 SpliceAI 和 Pangolin 来预测功能后果的变异，然后进行长读长 cDNA 测序以识别异常转录本。对于 120 个先前未解决的家族中的 8 个 （6%） ，BRCA1、PALB2 和 ATM 中罕见的深内含子变体会产生内含子伪外显子，这些伪外显子被剪接成转录本，导致过早截断。这些结果表明，长读长 DNA 和 cDNA 测序可以整合到变异发现中，并具有准确表征致病性变异的策略。