A Mouse Model of Schnyder Corneal Dystrophy with the N100S Point Mutation

Fei Dong,Christian A Combs,Christina Del Greco,Xueting Jin,Harrison Sciulli,Mones Abu-Asab,Ludovic Muller,Yueh-Chiang Hu,Shurong Wang,Winston W Kao,Amina S Woods,Michelle A Boettler,Howard S Kruth,Jianhua Zhang,Shelley N Jackson,Jayne S Weiss,Maria M Campos,Michael L Nickerson

doi:10.1038/s41598-018-28545-0

Abstract

Schnyder corneal dystrophy (SCD) is a rare autosomal dominant disease in humans, characterized by abnormal deposition of cholesterol and phospholipids in cornea caused by mutations in the UbiA prenyltransferase domain containing 1 (UBIAD1) gene. In this study, we generated a mouse line carrying Ubiad1 N100S point mutation using the CRISPR/Cas9 technique to investigate the pathogenesis of SCD. In vivo confocal microscopy revealed hyper-reflective dot-like deposits in the anterior cornea in heterozygotes and homozygotes. No significant change was found in corneal epithelial barrier function or wound healing. Electron microscopy revealed abnormal mitochondrial morphology in corneal epithelial, stromal, and endothelial cells. Mitochondrial DNA copy number assay showed 1.27 ± 0.07 fold change in homozygotes versus 0.98 ± 0.05 variation in wild type mice (P < 0.05). Lipidomic analysis indicated abnormal metabolism of glycerophosphoglycerols, a lipid class found in mitochondria. Four (34:1, 34:2, 36:2, and 44:8) of the 11 glycerophosphoglycerols species identified by mass spectrometry showed a significant increase in homozygous corneas compared with heterozygous and wild-type mouse corneas. Unexpectedly, we did not find a difference in the corneal cholesterol level between different genotypes by filipin staining or lipidomic analysis. The Ubiad1N100S mouse provides a promising animal model of SCD revealing that mitochondrial dysfunction is a prominent component of the disease. The different phenotype in human and mouse may due to difference in cholesterol metabolism between species.

Highlights

The gene UbiA Prenyltransferase Domain Containing 1 (UBIAD1), known as TERE1(transitional epithelial response protein 1), on human chromosome 1p36 locus is the causative gene of this corneal dystrophy[2,5,6]
Three sgRNAs were designed for Ubiad1N100S gene targeting and tested for the cleavage efficiencies in mK4 cells. sgRNA 3, with the highest cleavage efficiency, together with SpCas[9] mRNA and donor oligos, which harbored 5′-76 bp and 3′-60 bp homology arms, were injected to C57BL/6 N mouse zygotes to generate Ubiad1N100S knock in mice
A major finding in the Ubiad[1] mutant mouse that we report is the effect of the N100S mutation on mitochondrial morphology and biosynthesis

Summary

Introduction

The gene UbiA Prenyltransferase Domain Containing 1 (UBIAD1), known as TERE1(transitional epithelial response protein 1), on human chromosome 1p36 locus is the causative gene of this corneal dystrophy[2,5,6]. Studies show that UBIAD1 is a prenyltransferase participating in menaquione-4 (i.e., vitamin K2) synthesis and cholesterol metabolism[2,7]. It is an antioxidant enzyme regulating eNOS (endothelial nitric oxide synthase 3) activity[8]. Twenty five independent putative mutations have been reported p.A97T, p.G98S, p.N102S, Pharmacology, Louisiana State University School of Medicine, Louisiana State University Eye Center, Louisiana State. There is no SCD animal model available for researchers and clinicians to study this disease. We report an Ubiad[1] mutant mouse line that we created by the CRISPR/Cas[9] gene editing technique to carry a missense mutation N100S, corresponding to the human UBIAD1 N102S mutation

Methods

Results

Discussion

Conclusion