Long-read technologies unravel hidden inversion-duplication in a family with choroideremia

Abstract

Purpose : The lack of molecular diagnoses in rare genetic diseases can be explained by limitations of current standard genomic technologies. Upcoming long-read (sequencing) techniques have complementary strengths to overcome these limitations. By using optical mapping and long-read sequencing, we aimed to identify the pathogenic variant in a large family with X-linked choroideremia. In this family, aberrant splicing of exon 12 of the choroideremia gene CHM was detected in 2003, but the underlying genomic defect remained elusive.

Methods : We performed optical imaging followed by long-read whole genome sequencing to enable identification of a hidden structural variant in affected cases and carrier females in the studied family. This was followed by Sanger sequencing validation and segregation analysis. In silico analysis was performed to evaluate putative hair-pin formation as an underlying mechanism to exon 12 skipping in the mRNA.

Results : Optical mapping and long-read sequencing approaches now revealed an intragenic 1,752 bp inverted duplication including exon 12 and surrounding regions, located downstream of the wild-type copy of exon 12. Both breakpoint junctions were confirmed with Sanger sequencing, segregate with the X-linked inheritance in the family. The breakpoint junctions displayed sequence microhomology suggestive for an erroneous replication mechanism as the origin of the structural variant. The inverted duplication is predicted to result in hairpin-formation with the wild-type exon 12, which leads to skipping of this exon in the mature mRNA.

Conclusions : The identified inverted duplication is deemed the pathogenic cause of disease in this family. Our study shows that long-read sequencing and optical genome imaging techniques has significant potential for identification of structural variants in genetic diseases.

This is a 2021 ARVO Annual Meeting abstract.