Yang Yongping, Swaminathan Srividya, Martin Betty K, Sharan Shyam K
Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, 1050 Boyles Street, Frederick, MD 21702, USA.
Hum Mol Genet. 2003 Sep 1;12(17):2121-31. doi: 10.1093/hmg/ddg222. Epub 2003 Jul 8.
Numerous missense mutations in human BRCA1 gene have been linked to predisposition to breast cancer. However, the functional significance of the majority of these mutations remains unknown. We have examined the molecular basis for three such cancer-causing mutations. The first mutation, a T-->G transversion in codon 64, is predicted to change a conserved cysteine residue to glycine in the RING finger domain of the 1863 amino acid BRCA1 protein. Using a humanized mouse model we demonstrate that this missense mutation actually results in a functionally null protein. This striking result occurs because the single base alteration generates a new 5' splice site in exon 5 and also disrupts a putative exonic splicing enhancer motif. Consequently, the normal splice donor site is disrupted and an internal cryptic splice site is activated. This results in a 22-nucleotide deletion and the aberrant transcript is predicted to encode a severely truncated protein consisting of only 63 amino acids. To identify other missense mutations in BRCA1 that may result in aberrant splicing, we screened various mutations using the Genscan program. We demonstrate that at least two other missense mutations in codons 1495 and 1823 result in aberrant splicing due to the possible disruption of cis-acting splicing regulatory elements. In conclusion, our study demonstrates for the first time the application of a humanized mouse model for functional analysis of human mutations in mice and also shows the need for a careful examination of the functional consequences of single base alterations and single nucleotide polymorphisms identified in human disease-causing genes.
人类BRCA1基因中的众多错义突变已被证实与乳腺癌易感性相关。然而，这些突变中大多数的功能意义仍不明确。我们研究了三个此类致癌突变的分子基础。第一个突变是密码子64处的T→G颠换，预计会将1863个氨基酸的BRCA1蛋白的RING指结构域中一个保守的半胱氨酸残基变为甘氨酸。利用人源化小鼠模型，我们证明这种错义突变实际上导致了一种功能缺失的蛋白质。这一惊人结果的出现是因为单个碱基改变在第5外显子中产生了一个新的5'剪接位点，同时也破坏了一个假定的外显子剪接增强子基序。因此，正常的剪接供体位点被破坏，一个内部隐蔽剪接位点被激活。这导致了22个核苷酸的缺失，并且异常转录本预计编码一个仅由63个氨基酸组成的严重截短的蛋白质。为了鉴定BRCA1中其他可能导致异常剪接的错义突变，我们使用Genscan程序筛选了各种突变。我们证明，密码子1495和1823处的至少另外两个错义突变由于可能破坏顺式作用剪接调节元件而导致异常剪接。总之，我们的研究首次证明了人源化小鼠模型在小鼠中对人类突变进行功能分析的应用，同时也表明需要仔细研究在人类致病基因中鉴定出的单个碱基改变和单核苷酸多态性的功能后果。
