Novel Splice-Altering Variants in the CHM and CACNA1F Genes Causative of X-Linked Choroideremia and Cone Dystrophy

Background: An estimated 10-15% of all genetic diseases are attributable to variants in noncanonical splice sites, auxiliary splice sites and deep-intronic variants. Most of these unstudied variants are classified as variants of uncertain significance (VUS), which are not clinically actionable. This study investigated two novel splice-altering variants, CHM NM_000390.4:c.941-11T>G and CACNA1F NM_005183.4:c.2576+4_2576+5del implicated in choroideremia and cone dystrophy (COD), respectively, resulting in significant visual loss.

Methods: Next-generation sequencing was employed to identify the candidate variants in CHM and CACNA1F, which were confirmed using Sanger sequencing. Cascade analysis was undertaken when additional family members were available. Functional analysis was conducted by cloning genomic regions of interest into gateway expression vectors, creating variant and wildtype midigenes, which were subsequently transfected into HEK293 cells. RNA was harvested and amplified by RT-PCR to investigate the splicing profile for each variant compared to the wildtype. Novel variants were reclassified according to ACMG/AMP and ClinGen SVI guidelines.

Results: Midigene functional analysis confirmed that both variants disrupted splicing. The CHM NM_000390.4:c.941-11T>G variant caused exon 8 skipping, leading to a frameshift and the CACNA1F NM_005183.4:c.2576+4_2576+5del variant caused a multimodal splice defect leading to an in-frame insertion of seven amino acids and a frameshift. With this evidence, the former was upgraded to likely pathogenic and the latter to a hot VUS.

Conclusions: This study adds to the mutational spectrum of splicing defects implicated in retinal degenerations by identifying and characterising two novel variants in CHM and CACNA1F. Our results highlight the importance of conducting functional analysis to investigate the consequences of intronic splice-altering variants and the significance of reclassifying VUS to confirm a genetic diagnosis.