Human MutationRESEARCH ARTICLE Sequence Contexts That Determine the Pathogenicity of Base Substitutions at Position13 of Donor Splice-Sites

(50ss) are reported to induce aberrant splicing in some cases but not in others suggesting that the overall nucleotidic environment can dictate the extent to which 50ss are correctly selected. Functional studies of three variations identified in donor splice-sites of USH2A and PCDH15 genes sustain this assumption. To gain insights into this question, we compared the nucleotidic context of U2-dependent 50ss naturally deviated (13G,13C, or 13T) from the13A consensus with 50ss for which a 13 variation (A4G, A4C, or A4T) was shown to induce aberrant splicing. Statistical differences were found between the two datasets, highlighting the role of one peculiar position in each context (13G/14A;13C/!1G; and13T/!1G). We provided experimental support to the biostatistical results through the analysis of a series of artificial mutants in reporter minigenes. Moreover, different 50 end-mutated U1 snRNA expression plasmids were used to investigate the importance of the position 13 and of the two identified compensatory positions!1 and 14 in the recognition of 50ss by the U1 snRNP. Overall, our findings establish general properties useful to molecular geneticists to identify nucleotide substitutions at position 13 that are more likely to alter splicing