Caligo Maria Adelaide, Bonatti Fabrizia, Guidugli Lucia, Aretini Paolo, Galli Alvaro
Sezione Genetica Oncologica Divisione di Patologia Dipartimento di Oncologia Università di Pisa, Pisa, Italy.
Hum Mutat. 2009 Jan;30(1):123-33. doi: 10.1002/humu.20817.
The BRCA1 tumor suppressor gene is found mutated in familial breast cancer. Although many of the mutations are clearly pathological because they give rise to truncated proteins, several missense variants of uncertain pathological consequences have been identified. A novel functional assay to screen for BRCA1 missense variants in a simple genetic system could be very useful for the identification of potentially deleterious mutations. By using two prediction computer programs, Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen), seven nonsynonymous missense BRCA1 variants likely disrupting the gene function were selected as potentially deleterious. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) was used to test these cancer-related missense mutations for their ability to affect cell growth and homologous recombination (HR) at the HIS3 and ADE2 loci. The variants localized in the BRCA1 C-Terminus (BRCT) domain did not show any growth inhibition when overexpressed in agreement with previous results. Overexpression of either wild-type BRCA1 or two neutral missense variants did not increase yeast HR but when cancer-related variants were overexpressed a significant increase in recombination was observed. Results clearly showed that this genetic system can be useful to discriminate between neutral and deleterious BRCA1 missense variants.
BRCA1肿瘤抑制基因在家族性乳腺癌中被发现发生突变。尽管许多突变明显具有致病性，因为它们会产生截短的蛋白质，但也已鉴定出一些病理后果不确定的错义变体。在一个简单的遗传系统中筛选BRCA1错义变体的新型功能测定法，对于识别潜在有害突变可能非常有用。通过使用两个预测计算机程序，即从耐受中筛选不耐受（SIFT）和多态性表型分析（PolyPhen），选择了七个可能破坏基因功能的非同义BRCA1错义变体作为潜在有害变体。利用芽殖酵母酿酒酵母来测试这些与癌症相关的错义突变影响HIS3和ADE2位点细胞生长和同源重组（HR）的能力。定位于BRCA1 C末端（BRCT）结构域的变体在过表达时未显示出任何生长抑制，这与先前的结果一致。野生型BRCA1或两个中性错义变体的过表达均未增加酵母的HR，但当与癌症相关的变体过表达时，观察到重组显著增加。结果清楚地表明，这个遗传系统可用于区分BRCA1错义变体的中性和有害性。
