All single nucleotide substitutions and single nucleotide deletions recorded in the database have been analyzed with splicing prediction tools available on the web. Two tools have been used, NNSPLICE 0.9 and HSF V2.3.

Only splice sites have been analyzed (enhencers or branch sites have not been analyzed). Please refer to the respective web site tools for explanations on the algorithms used by these tools.

The following annotations are available in the display table (use the 'Mutation validation' or 'Function pattern' options):
-  Mutation: Description of the mutation at the coding level.
-  Site type: Nature of splice site (5' acceptor or 3' donor).
-  p53 site: Actual p53 splice sites used for generating the full-lenght protein.
-  WT score: Score of the identified splice site in the wild-type sequence.
-  MUT score: Score of the identified splice site in the mutated sequence.
-  Variation: Interpreted score change (see below).
-  Source: Name of the program that has identified the splice site.

Mutations (in the format: c.524G>A) were submitted to HSF web site and analyzed with the option "Analyze mutation(s)" with default settings and with TP53 gene as reference sequence. Predictions results for splice sites were retrieved. The sites recognized by HSF have the following structure:
-  Donor site: 3 bases in the exons and 6 bases in the intron, ie CTGgtaagg
-  Acceptor site: 2 bases in the exons and 12 bases in the intron, ie acccatctacagTC
The scores generated by HSF range from 0 to 100, 100 corresponding to a perfect match with a consensus splice site. A threshold of 65 is used by HSF to filter results, so that scores are retrieved only if a motif with a score>65 has been found either in the wild-type or mutant sequence.

The following rules have been then applied to annotate the "Variation" column:

-  No significant change: no splice site motif with a score>65 was found at the position of the mutation in the WT or MUT sequence, or the score found for the MUT motif was identical to the WT, or both WT and MUT motifs have a score>65;
-  New site: the mutation created a splice motif not present in the wt sequence (WT motif score<65 and MUT motif score>65);
-  Site broken: the mutation removed a splice motif that was identified in the wt sequence (WT motif score>65 and MUT motif score<65).

The input for NNSPLICE is a sequence in FASTA format. Sequences for all mutations have thus been generated from our reference sequence and the WT and MUT sequences have been submitted to NNSPLICE web site. The sites recognized by HSF have the following structure:
-  Donor site: 7 bases in the exons and 8 bases in the intron, ie AAGCGAGgtaagcaa
-  Acceptor site: 20 bases in the exons and 21 bases in the intron, ie tcatcttctgtcccttcccagAAAACCTACCAGGGCAGCTA
Splice motifs found in these sequences were retrieved, along with their scores. The scores generated by NNSPLICE range from 0 to 1, 1 corresponding to a perfect match with a consensus splice site. A threshold of 0.4 is used by NNSPLICE to filter results, so that scores are retrieved only if a motif with a score>0.4 has been found either in the wild-type or mutant sequence.

The same rules as HSF have then been applied to annotate the "Variation" column:

-  No significant change: no splice site motif with a score>0.4 was found at the position of the mutation in the WT or MUT sequence, or the score found for the MUT motif was identical to the WT, or both WT and MUT motifs have a score>0.4;
-  New site: the mutation created a splice motif not present in the wt sequence (WT motif score<0.4 and MUT motif score>0.4);
-  Site broken: the mutation removed a splice motif that was identified in the wt sequence (WT motif score>0.4 and MUT motif score<0.4).