Weak definition of IKBKAP exon 20 leads to aberrant splicing in familial dysautonomia

El Chérif Ibrahim, Matthew M. Hims, Noam Shomron, Christopher B. Burge, Susan A. Slaugenhaupt, Robin Reed

Research output: Contribution to journalArticlepeer-review


Splicing mutations that lead to devastating genetic diseases are often located in nonconserved or weakly conserved sequences that normally do not affect splicing. Thus, the underlying reason for the splicing defect is not immediately obvious. An example of this phenomenon is observed in the neurodevelopmental disease familial dysautonomia (FD), which is caused by a single-base change in the 5′ splice site (5′ss) of intron 20 in the IKBKAP gene (c.2204+6T>C). This mutation, which is in the sixth position of the intron and results in exon 20 skipping, has no phenotype in many other introns. To determine why the position 6 mutation causes aberrant splicing only in certain cases, we first used an in silico approach to identify potential sequences involved in exon 20 skipping. Computational analyses of the exon 20 5′ss itself predicted that this nine-nucleotide splicing signal, even when it contains the T>C mutation, is not sufficiently weak to explain the FD phenotype. However, the computational analysis predicted that both the upstream 3′ splice site (3′ss) and exon 20 contain weak splicing signals, indicating that the FD 5′ss, together with the surrounding splicing signals, are not adequate for defining exon 20. These in silico predictions were corroborated using IKBKAP minigenes in a new rapid and simple in vitro coupled RNA polymerase (RNAP) II transcription/splicing assay. Finally, the weak splicing signals that flank the T>C mutation were validated as the underlying cause of familial dysautonomia in vivo using transient transfection assays. Together, our study demonstrates the general utility of combining in silico data with an in vitro RNAP II transcription/splicing system for rapidly identifying critical sequences that underlie the numerous splicing diseases caused by otherwise silent mutations.

Original languageEnglish
Pages (from-to)41-53
Number of pages13
JournalHuman Mutation
Issue number1
StatePublished - Jan 2007
Externally publishedYes


  • 5′ splice site
  • Exon skipping
  • Familial dysmitonomia
  • RNA splicing


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