Petersen Annabeth Høgh, Aagaard Mads Malik, Nielsen Henriette Roed, Steffensen Karina Dahl, Waldstrøm Marianne, Bojesen Anders
Department of Clinical Genetics, Vejle Hospital, Lillebaelt Hospital, Vejle, Denmark.
Department of Clinical Oncology, Vejle Hospital, Lillebaelt Hospital, Vejle, Denmark.
Eur J Hum Genet. 2016 Aug;24(8):1104-11. doi: 10.1038/ejhg.2015.268. Epub 2016 Jan 6.
Accurate estimation of cancer risk in HBOC families often requires BRCA1/2 testing, but this may be impossible in deceased family members. Previous, testing archival formalin-fixed, paraffin-embedded (FFPE) tissue for germline BRCA1/2 variants was unsuccessful, except for the Jewish founder mutations. A high-throughput method to systematically test for variants in all coding regions of BRCA1/2 in archival FFPE samples of non-tumor tissue is described, using HaloPlex target enrichment and next-generation sequencing. In a validation study, correct identification of variants or wild-type was possible in 25 out of 30 (83%) FFPE samples (age range 1-14 years), with a known variant status in BRCA1/2. No false positive was found. Unsuccessful identification was due to highly degraded DNA or presence of large intragenic deletions. In clinical use, a total of 201 FFPE samples (aged 0-43 years) were processed. Thirty-six samples were rejected because of highly degraded DNA or failed library preparation. Fifteen samples were investigated to search for a known variant. In the remaining 150 samples (aged 0-38 years), three variants known to affect function and one variant likely to affect function in BRCA1, six variants known to affect function and one variant likely to affect function in BRCA2, as well as four variants of unknown significance (VUS) in BRCA1 and three VUS in BRCA2 were discovered. It is now possible to test for germline BRCA1/2 variants in deceased persons, using archival FFPE samples from non-tumor tissue. Accurate genetic counseling is achievable in families where variant testing would otherwise be impossible.
准确估计HBOC家族的癌症风险通常需要进行BRCA1/2检测，但对于已故家族成员而言这可能无法实现。此前，除了犹太始祖突变外，对存档的福尔马林固定石蜡包埋（FFPE）组织进行种系BRCA1/2变异检测均未成功。本文描述了一种高通量方法，利用HaloPlex目标富集和新一代测序技术，系统检测非肿瘤组织存档FFPE样本中BRCA1/2所有编码区的变异。在一项验证研究中，30份FFPE样本（年龄范围1 - 14岁）中有25份（83%）能够正确鉴定出变异或野生型，这些样本的BRCA1/2变异状态已知，且未发现假阳性结果。鉴定失败是由于DNA高度降解或存在大片段基因内缺失。在临床应用中，共处理了201份FFPE样本（年龄0 - 43岁）。36份样本因DNA高度降解或文库制备失败而被拒收。对15份样本进行检测以寻找已知变异。在其余150份样本（年龄0 - 38岁）中，发现了BRCA1中3个已知影响功能的变异和1个可能影响功能的变异、BRCA2中6个已知影响功能的变异和1个可能影响功能的变异，以及BRCA1中4个意义未明的变异（VUS）和BRCA2中3个VUS。现在可以利用非肿瘤组织的存档FFPE样本对已故人员进行种系BRCA1/2变异检测。在那些否则无法进行变异检测的家族中，可以实现准确的遗传咨询。
