The resolution of the paternal allele was hindered by the fact that the region spanning intron 20 to exon 29 of OTOA displays very high sequence identity with exons 1 to 9 of the OTOAP1 pseudogene (NR_003676.3) and therefore lacks MLPA probes. Upon visual inspection of the reads aligning to exon 22 of OTOA, we hypothesized the presence on the paternal chromosome of either a small deletion of exon 22 of OTOA or of a conversion of the wild-type sequence of OTOA by OTOAP1.
A 13kb PCR product specific to the paternally inherited OTOA allele was obtained using the proband’s DNA and primer #1 and #2; primer #1 being specific to OTOA and primer #2 to both OTOA and OTOAP1 (Fig. 1C). This PCR product was then Sanger sequenced using a set of internal primers listed in Supplementary Table 1 (Fig. 1C).
Taken together the sequencing results showed that the paternal OTOA allele was not carrying a deletion of exon 22 but underwent pseudogene-mediated gene conversion. This conversion event introduced several sequence variants contributed by OTOAP1 into OTOA including among others a pathogenic premature stop codon (p.Glu787*) (Fig. 1E).