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 CEP78 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 CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 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 CEP78 expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting CEP78 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 CEP78 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 CEP78 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.

StrVCTVRE: A supervised learning method to predict the pathogenicity of human genome structural variants

“…Best practices for SV prioritization are evolving, and generally mirror steps used to prioritize SNVs. Few SV-tailored impact predictors have been developed, but a small number of published studies have focused on identifying pathogenic SVs from WES 10 , 11 and WGS 7 , 12 , 13 and have identified a handful of important steps. Removing low-quality SV calls is essential, as short-read SV callers rarely achieve precision above 80% for deletions and 50% for duplications, even at low recall.…”

Loss-of-function mutations in CEP78 cause male infertility in humans and mice

“…CEP78 is a known causative gene for CRDHL syndrome, which is characterized by cone-rod dystrophy and sensorineural hearing loss, with relatively late onset of both ocular and auditory impairments ( 19 – 22 ). Thus, our proband showed defects in hearing and vision.…”

“…To date, several inactivating variants, a missense variant, and two complex structural variants in CEP78 have been identified in individuals with autosomal recessive CRDHL syndrome, a rare syndromic inherited retinal disease accompanied by sensorineural hearing loss (19)(20)(21)(22). In addition, two splicing mutations in CEP78 were identified in two independent families with a mild degree of Usher syndrome, a genetic disorder characterized by the combination of retinitis pigmentosa and sensorineural hearing loss (50).…”

“…Biallelic inactivating variants in CEP78 have been linked to autosomal recessive cone-rod dystrophy with hearing loss (CRDHL) syndrome [OMIM (Mendelian Inheritance in Man) no. 617236] in humans (19)(20)(21)(22). However, the biological function of CEP78 in male reproduction remains poorly characterized.…”

EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders

“…For example, the recent elucidation of DYNC2H1 as a cause of inherited ophthalmic conditions was not captured in our initial curation process but can be subsequently included in EyeG2P analysis through addition of a single data line to the released EyeG2P datafile. 25 Our ability to detect disease-causing genomic variants from high-throughput sequencing datasets has expanded in recent years to include complex structural variants, 26,27 exonic deletions and duplications, 28,29 deeply intronic variants causing aberrant splicing, [30][31][32][33] variants in regulatory regions [34][35][36] and complex alleles comprised of combinations of genomic variants common in the general population. 37,38 Here we found that, in addition to characterizing novel exonic variants, EyeG2P is capable of prioritizing these diverse types of disease-causing variation, achieving 99.5% sensitivity in comparison to routine analytical approaches (Figure 1).…”