Extending the Genotypes and Phenotypes of C1QTNF5-Associated Retinal Disease

Background: Late-Onset Retinal Degeneration (L-ORD) is an inherited retinal dystrophy (IRD) that typically manifests with delayed dark adaptation by the fifth decade of life and progresses to initial central visual impairment after age 60 years. Here, we describe the clinical and molecular characterisation of two C1QTNF5 missense variants identified in individuals with IRD that do not share the characteristic retinal phenotype of L-ORD and exhibit variable age of onset and disease severity. Methods: Probands from two unrelated families underwent genetic testing and comprehensive ophthalmic phenotyping, including multimodal retinal imaging and electrophysiological assessment. This allowed comparison to the previously reported ophthalmic phenotype in two additional families for which the genetic cause of IRD had been previously reported. Genetic analysis was performed using next-generation sequencing, with variants classified according to ACMG/AMP guidelines. In silico analyses assessed evolutionary conservation, predicted pathogenicity, and structural consequences. To experimentally assess variant effects, wild-type and mutant C1QTNF5 constructs were expressed in hTERT-RPE1 cells, and protein folding, stability, and secretion were evaluated. Results: A de novo heterozygous variant, c.548G>A p.(G183E) in C1QTNF5 in a proband, was classified as likely pathogenic. The genetic cause of IRD in the other unrelated probands was C1QTNF5 c.538C>G, p.(Q180E), a known pathogenic variant. The missense variants affect highly conserved residues and are absent from population databases. Structural modelling of both variants predicted destabilisation of C1QTNF5 for both variants, although significantly less severe than for previously identified pathogenic L-ORD variants. In vitro analyses revealed reduced stability of both mutant proteins expressed alone, but when co-expressed with wild-type (WT) C1QTNF5, differences between these variants were observed. In contrast to L-ORD variants, C1QTNF5 containing p.G183E or p.Q180E, could multimerise, generating the expected ratio of trimer and high molecular weight C1QTNF5 multimers. C1QTNF5 p.Q180E/WT co-assembly destabilised the protein and decreased the amount of total C1QTNF5 secreted, but p.G183E was secreted at an equivalent level to WT C1QTNF5. Conclusions: These findings expand the variant and mechanistic spectrum of C1QTNF5-associated retinal disease and highlight C1QTNF5 protein misfolding as a key pathogenic mechanism. Integrating clinical phenotyping with structural and functional analyses is critical for accurate variant interpretation and molecular diagnosis in C1QTNF5-associated retinal disease.