Barrois M, Bièche I, Mazoyer S, Champème M-H, Bressac-de Paillerets B, Lidereau R
Service de Génétique, Institut Gustave Roussy, Villejuif, France.
Clin Genet. 2004 Feb;65(2):131-6. doi: 10.1111/j.0009-9163.2004.00200.x.
BRCA1 and BRCA2 germline mutations, mainly point mutations and other small alterations, are responsible for most hereditary cases of breast-ovarian cancer. However, the observed frequency of BRCA1 alterations is lower than that predicted by linkage analysis. Several large BRCA1 rearrangements have been identified with a variety of technical approaches in some families. We have developed a gene dosage assay based on real-time quantitative PCR and used it to extensively analyze 91 French families of breast-ovarian cancer in which no BRCA1 or BRCA2 point mutations was identified. This gene dosage method calculates the copy number of each BRCA1 exon to readily detect one, two, and three or more copies of BRCA1 target exons. In the series of 91 families at high risk of carrying BRCA1 mutations, we detected seven large rearrangements of the BRCA1 gene by using this real-time PCR approach. This simple, rapid, and semiautomated real-time quantitative polymerase chain reaction (PCR) assay is a promising alternative technique to Southern blot, bar code analysis on combed DNA, quantitative multiplex PCR of short fluorescent fragments, and cDNA length analysis for the detection of large rearrangements. Therefore, this technique should be considered as a powerful diagnostic method for breast/ovarian cancer susceptibility in clinical and research genetic surveys.
BRCA1和BRCA2种系突变，主要是点突变和其他小的改变，是大多数遗传性乳腺-卵巢癌病例的病因。然而，观察到的BRCA1改变的频率低于连锁分析预测的频率。在一些家族中，已经通过多种技术方法鉴定出了几种大型的BRCA1重排。我们开发了一种基于实时定量PCR的基因剂量测定法，并将其用于广泛分析91个未鉴定出BRCA1或BRCA2点突变的法国乳腺-卵巢癌家族。这种基因剂量方法计算每个BRCA1外显子的拷贝数，以轻松检测BRCA1目标外显子的一个、两个和三个或更多个拷贝。在这91个携带BRCA1突变高风险家族的系列研究中，我们使用这种实时PCR方法检测到了7个BRCA1基因的大型重排。这种简单、快速且半自动的实时定量聚合酶链反应（PCR）测定法是一种很有前景的替代技术，可替代Southern印迹法、梳理DNA上的条形码分析、短荧光片段的定量多重PCR以及用于检测大型重排的cDNA长度分析。因此，在临床和研究基因调查中，该技术应被视为一种用于乳腺/卵巢癌易感性的强大诊断方法。
