Abstract
Purpose :
Dehydrodolichyl diphosphate synthase (DHDDS) is ubiquitously expressed and serves an essential function throughout the body, yet certain defects in the gene cause non-syndromic retinitis pigmentosa (RP59). To understand the basis for this retina selectivity, we generated and characterized two novel Dhdds knock-in mouse models: DhddsT206A/T206A and DhddsT206A/K42E.
Methods :
Heterozygous (DhddsT206A/+) knock-in (KI) mice were generated on a C57Bl/6J background using CRISPR/Cas9 technology (UAB Transgenic & Genetically Engineered Models Core). DhddsT206A/+ littermates were crossed to generate Dhdds+/+, DhddsT206A/+, and DhddsT206A/T206A mice, and with DhddsK42E/K42E to generate DhddsT206A/K42E mice (to at least 3rd (F3) generation). All mutations were verified by PCR and sequence analysis. Phenotypes were assessed by SD-OCT and electroretinography (ERG). Retinal structure was assessed for DhddsT206A/T206A, DhddsT206A/+, DhddsT206A/K42E, and wild type (WT) mice (N=3-5) at PN 1 and 3 mo., while ERG was performed at PN 3 mo. Statistical analysis: Student t-test, with p<0.05 as significance threshold.
Results :
SD-OCT showed no significant differences in retinal morphology, comparing mutant vs. WT mice. Outer nuclear layer (ONL) thickness (µm; mean±S.D.): DhddsT206A/T206A (55±5); DhddsT206A/+ (54±6); DhddsT206A/K42E (56±5); vs. WT (55±7). Total retinal thickness (µm): DhddsT206A/T206A (222±8); DhddsT206A/+ (220±8); DhddsT206A/K42E (220±9); vs. WT (222±6). DhddsT206A/+ ERG maximum response amplitudes at saturation were not significantly different from WT values under dark-adapted (scotopic) or light-adapted (photopic) conditions. However, photopic ERG amplitude of DhddsT206A/K42E mice were significantly different from WT values, and trending lower for DhddsT206A/T206A mice. Additionally, scotopic b-/a-wave ratios (values in parentheses) of DhddsT206A/T206A (1.2:1) and DhddsT206A/K42E (1.3:1), vs. WT (2:1) mice were significantly diminished.
Conclusions :
Homozygous (T206A/T206A) and compound heterozygous (K42E/T206A) Dhdds knock-in mouse models of RP59 exhibit negative ERGs under light- and dark-adapted conditions, indicative of defective photoreceptor-to-bipolar cell synaptic transmission. There may be common retina-specific target(s) that could explain the lack of systemic effects associated with DHDDS mutations in RP59.
This is a 2021 ARVO Annual Meeting abstract.