Splicing mutation associated with Rett syndrome and an experimental approach for genetic diagnosis.

Around 80% of Rett syndrome (RS) cases have a mutation or deletion within the coding sequence of the MeCP2 gene. The other RS patients remain genetically undiagnosed. A significant fraction (10-15%) of disease-causing mutations in humans, affect pre-mRNA splicing. Two potential splice mutations were found in the MeCP2 gene in RS patients, however it was not clear whether these mutations in fact interfere with splicing and consequently cause RS. One such mutation is a deletion of the GT dinucleotide at the 5' donor splice site of exon 1 and the other a deletion of the T nucleotide in the polypyrimidine tract (PPT) of intron 3. Here we experimentally assess the effects exerted by these mutations on the expression of MeCP2 in patients' blood samples and on splicing of the MeCP2 transcript using a hybrid minigene in transient transfection experiments. The results revealed that the Delta T mutation in the PPT is a benign polymorphism and that the GT deletion in intron 1 is a bona fide splicing mutation that causes a complete skipping of exon 1 in the minigene transfection experiment. This explains the observed complete elimination of the MeCP2 message and protein in the lymphoblast clones of the RS patient that carry the mutation on the active X. An analysis of the MeCP2 transcript and protein production in lymphoblast clones, as described here, can be used to confirm clinically diagnosed RS patients with no mutation in the MeCP2 coding sequence. This will enable RS diagnosis without specifically identifying a mutation.