史上最精准基因测序:原来健康人也普遍存在癌症相关突变
医脉通 · 2016/05/10
双重测序技术可以检测到极稀少的突变,理论上对一个细胞的全基因组进行精确测序而不出现一个错误。使用双重测序技术发现TP53突变在恶性肿瘤中普遍存在。


二代测序(NGS)技术在基因测序深度和测序精度方面突飞猛进,为精准医学的发展提供了重要的工具支持。2012年,美国华盛顿大学的研究人员开发了一种新型的基因测序技术,该技术被命名为[Duplex sequencing](暂译为双重测序)。顾名思义,双重测序就是对DNA的双链分别进行独立的标记和测序,然后比对两条链中每一段的序列。由于DNA双链的互补性,真实的突变应同时发生于两条链的同一位置,而某个突变仅存在于一条链则被认为是测序错误,这样就能显著地减少假阳性突变的出现,并将测序精度推向很高的水平。

双重测序技术可以检测到极稀少的突变,理论上对一个细胞的全基因组进行精确测序而不出现一个错误。

华盛顿大学的这项双重测序技术近几年不断完善,并在PNAS、Nature Protocols等杂志发表了一系列成果,检测精度目前无人能及。研究团队在卵巢癌探索了双重测序的检测能力,旨在确认其能否检测到高级别浆液性卵巢癌(HGSOC)患者腹水中极少量的癌细胞,这也是该技术首次应用于人类肿瘤组织。研究结果2016年5月5日在线发表于Proceedings of the National Academy of Sciences(PNAS)杂志。

该研究使用双重测序技术分别在17例HGSOC女性患者和20例健康女性的腹腔液样本中检测TP53突变。P53是抑癌基因,该基因突变后导致所编码的蛋白丧失重要的细胞增殖调控作用。TP53突变在恶性肿瘤中普遍存在,HGSOC中该基因突变率高达96%。


(图片来自研究者之一Elizabeth Swisher,深棕色染色代表HGSOC细胞中突变型TP53表达蛋白)

◆对于HGSOC患者,双重测序在16例腹腔液中检测到TP53突变,占94%(检测精度为1突变/24736基因组)。

◆对于所有参与者,双重测序在35例腹腔液标本中都检测到了极低频率的TP53突变,而卵巢癌患者中TP53突变丰度高于正常健康人。

◆双重测序在15例健康女性血液中也检测到低频率TP53突变,可能与衰老相关。

研究者表示,下一步将扩大参与者人群,探究低频基因突变在癌症和衰老中的特点。

结语

该研究证实了双重测序对极少量肿瘤细胞的强大检测能力,也揭示了正常组织普遍存在极低频率的TP53突变,说明当检测精度足够高的时候,几乎所有人体内都能发现癌症相关基因突变。

另一方面,这项研究对精准医学提出了新的问题。如果临床医生试图通过基因测序发现肿瘤,那么如何正确识别正常组织中普遍存在的低频率癌症相关突变。未来可能需要建立癌症基因突变负荷标准或门槛,将癌变从正常的身体机能退化中鉴别出来。

信源:

1.Cancer-like mutations prevalent in apparently healthy group. hsnewsbeat.uw.edu.May 2, 2016.

2.Jeffrey D. Krimmel et al.Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues. Proc Natl Acad Sci.May 5,2016

3.Duplex-sequencing method could lead to better cancer detection and treatment. washington.edu.Sep 28,2012.

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  • Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues

    Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.

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