Whole USH2A Gene Sequencing Identifies Several New Deep Intronic Mutations

ABSTRACT Deep intronic mutations leading to pseudoexon (PE) insertions are underestimated and most of these splicing alterations have been identified by transcript analysis, for instance, the first deep intronic mutation in USH2A, the gene most frequently involved in Usher syndrome type II (USH2). Unfortunately, analyzing USH2A transcripts is challenging and for 1.8%–19% of USH2 individuals carrying a single USH2A recessive mutation, a second mutation is yet to be identified. We have developed and validated a DNA next‐generation sequencing approach to identify deep intronic variants in USH2A and evaluated their consequences on splicing. Three distinct novel deep intronic mutations have been identified. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. We present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. Moreover, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease‐linked gene. Finally, an antisense morpholino oligonucleotide tested in vitro for its ability to restore splicing caused by the c.9959‐4159A>G mutation provided high inhibition rates, which are indicative of its potential for molecular therapy. We present a strategy to identify deep intronic mutations, based on next‐generation sequencing of the complete gene of interest, prediction of splicing alterations and minigene assays. This approach is independent of the size of the gene and does not require RNA. After validation on a known variant leading to the insertion of a pseudoexon, the approach was applied to five patients carrying a monoallelic mutation in the 800 kb USH2A gene; three novel deep intronic disease‐causing variants were identified