Characterization of cone structure and function in a mouse model for blue cone monochromacy patients with a C203R mutation

Abstract

Purpose : Blue cone monochromacy (BCM) is a severe vision disorder caused by severely reduced or absence of function in L- and M-cones. Presence of a C203R missense mutation in a single OPN1LW/MW hybrid gene or in multiple OPN1LW/MW genes is the most common cause of BCM. In this study, we seek to dissect the disease mechanism of a C203R mutation through characterizing the structure and function of cone photoreceptors in a knock-in mouse model carrying a corresponding mutation.

Methods : Opn1mw^C198R (OPN1L/MW^C203R equivalent) knock-in mice were generated using CRISPR/Cas9 genome editing technology and were bred into an Opn1sw^-/- background to eliminate interference from endogenous S-opsin. Retinal whole mounts of Opn1mw^C198R mice were labeled with peanut agglutinin (PNA) to assess cone viability at 1, 3, 6, and 9 months of age. Quantitative RT-PCR was used to evaluate mRNA levels of Opn1mw^C198R at P5, P15, and P30. In tandem, cross-sections from Opn1mw^C198R retinas were examined by immunohistochemistry (IHC) using antibodies against L/M-opsin, PDE6α’, and GNAT2. Finally, M-cone mediated retinal function was analyzed at P30 by electroretinography (ERG). Age-matched C57BL/6J mice were used as controls.

Results : Ventral and dorsal regions of Opn1mw^C198R retina display significantly reduced cone viability by 3 months and cones continue to degenerate with age. By 6 months, Opn1mw^C198R cones in the dorsal and ventral regions of the retina are reduced by 60.0% and 43.9%, respectively, compared to those at 1 month of age (P <0.05). While qRT-PCR shows that Opn1mw mRNA is comparable between WT and mutant at P5, mutant mRNA levels drop 44% by P30 (P<0.05). Surprisingly, OPN1MW^C198R protein was not detected by IHC at P5, P15, or P30. GNAT2 staining appeared brighter at P5 compared to WT, but was mislocalized to the cone cell body and inner segment. Furthermore, GNAT2 staining dissipates by P15 and P30, suggesting cone outer segments are absent or significantly shortened in Opn1mw^C198R mice. Moreover, ERG demonstrates that Opn1mw^C198R eyes have no M-cone mediated retinal function at P30.

Conclusions : Cone structure and function in Opn1mw^C198R mice appear to phenocopy BCM patients with C203R mutations, demonstrating that this model can be adequately used for characterization of the disease mechanism behind a C203R mutation and in development of therapy.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.