Long-read sequencing to unravel complex structural variants of CEP78 leading to Cone-Rod Dystrophy and Hearing Loss

Abstract

Purpose : Inactivating sequence variants as well as a unique missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive Cone-Rod Dystrophy with Hearing Loss (CRDHL), a rare syndromic inherited retinal disease (IRD) clearly distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs and characterization of their underlying mechanisms.

Methods : Approaches used, for SVs identification, are shallow whole genome sequencing (sWGS) combined with quantitative PCR (qPCR) and long-range PCR, or ExomeDepth analysis on whole exome sequencing data. Targeted or whole genome Nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SV cases, the effect of the SVs on CEP78 expression was assessed using qPCR on patient-derived RNA. Data mining of human single cell (sc), bulk retinal and cochlear transcriptional datasets was used to further characterize the CEP78 expression profile.

Results : Apart from two novel canonical CEP78 splice variants and a frameshifting variant, three SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235kb and a partial gene deletion of 12kb in heterozygous and homozygous state respectively. Assessment of the molecular consequences of the SVs on patient’s materials displayed a loss-of-function effect. Delineation and characterization of the 12kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive for a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in three cases of Belgian and British origin respectively. The novel 235kb deletion was delineated using whole genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based mechanism behind CEP78 SV formation. Moreover, we correlated CEP78 sc expression with the phenotypic presentation of CRDHL.

Conclusions : Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic work-up of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole genome LRS in identifying and characterizing suspected complex SVs in patients with IRD.

This is a 2021 ARVO Annual Meeting abstract.