Elucidation of the molecular mechanism that allows functional splicing at human mutant +1G>T splice donor sites

实际上，人类5 ‘剪接位点（5 ’ s）的专性GT二核苷酸的所有突变都会导致剪接异常，因此被归类为致病性。然而，我们最近在来自三个家系的9例范可尼贫血（FA）患者中发现，范可尼贫血C （FANCC）基因外显子2的典型5 ' s上发生了+1G>T种系突变，令人惊讶地导致正确剪接转录本，尽管剪接水平较低。我们的结论是，这些转录本和检测到的正常翻译后加工FANCD2蛋白的微小水平可能有助于这些患者较轻的临床表现。受影响的FANCC 5’s的一个显著特征是其高内在强度，在我们的代表性数据集中，来自注释的本构剪接的人类外显子的所有剪接供体位点中，其排名为92.3个百分点。虽然在人类基因突变数据库（HGMD）中已经记录到+1G b> T突变的总体频率很高，但不知道有多少具有相当高内在强度的5 ' s也允许正确的5 ' s识别。通过体外和体内剪接分析，同时分析和比较TT剪接的例子，将获得对5 ' s识别的见解。我们将重点关注剪接供体位点及其周围的序列要求，以及可能参与TT剪接位点识别的蛋白质因子的鉴定。

Virtually all of the mutations at the obligate GT dinucleotide of a human 5’ splice site (5’ss) cause aberrant splicing and thus are classified as pathogenic. However, we have recently identified in nine Fanconi anemia (FA) patients from three pedigrees a +1G>T germline mutation in the canonical 5’ss of exon 2 of the Fanconi anemia C (FANCC) gene surprisingly resulting in correctly spliced transcripts albeit at a low level. We concluded that these transcripts and the detected minute levels of normal post-translationally processed FANCD2 protein might contribute to the milder clinical manifestations of the disease in these patients. One striking feature of the affected FANCC 5’ss was its high intrinsic strength which ranked at the 92.3 percentile of all splice donor sites in our representative data set from annotated constitutively spliced human exons. Although a +1G>T mutation has been documented with an overall high frequency within the Human Gene Mutation Database (HGMD), it is not known how many of the 5’ss with a comparable high intrinsic strength also allows correct 5’ss recognition. Insights into 5’ss recognition will be gained by simultaneously analyzing and comparing examples of TT splicing by in vitro and in vivo splicing analyses. We will focus on sequence requirements at and around the splice donor site as well on the identification of protein factors possibly involved in TT splice site recognition.