Cis ‐ and Trans ‐Regulatory Factors Independently Shape Phenotypic Heterogeneity of Retinitis Pigmentosa

ABSTRACT Retinitis pigmentosa (RP) is a group of retinal inherited diseases characterized by substantial clinical heterogeneity, even among individuals carrying the same pathogenic mutation. In this study, we identified two distinct genetic mechanisms influencing RP severity using a transgenic zebrafish model expressing the human rhodopsin S334X mutation. The first mechanism is tightly linked to RP onset and results in a benign phenotype. Whole‐genome sequencing revealed a 3‐base‐pair insertion upstream of the transgene, specific to the benign line, acting as a cis ‐regulatory variant that suppresses transgene expression, as validated by RT‐qPCR, Western blot and luciferase assays. The second mechanism operates independently of RP onset and modifies the benign phenotype toward a more severe presentation. This segregation was observed in approximately half of the offspring derived from specific individuals within the S334X‐benign line, consistent with a Mendelian ratio, suggesting a dominant trans ‐acting factor. Together, these findings provide the first in vivo evidence that both cis ‐ and trans ‐regulatory elements independently and cooperatively modulate RP phenotypes within a shared genetic background. They clearly establish the existence of genetic modifiers as key contributors to disease variability, offering new directions for personalized RP management.