Long-Read Sequencing to Unravel Complex Structural Variants of CEP78 Leading to Cone-Rod Dystrophy and Hearing Loss

Abstract

Inactivating variants as well as a missense variant in the centrosomal <i>CEP78</i> gene have been identified in autosomal recessive cone-rod dystrophy with hearing loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating <i>CEP78</i> has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the <i>CEP78</i> region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole-genome sequencing (sWGS) combined with quantitative polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) data. Targeted or whole-genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on <i>CEP78</i> expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical <i>CEP78</i> splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting <i>CEP78</i> were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a 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 15-kb deletion using targeted LRS revealed the previously described complex <i>CEP78</i> SV, suggestive of a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235-kb deletion was delineated using whole-genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the <i>CEP78</i> locus is prone to distinct SVs and that SV analysis should be considered in a genetic workup of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole-genome LRS in identifying and characterizing complex SVs in patients with ocular diseases.