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

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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.