Development of a rapid and cost-effective CRISPR-based molecular detection platform for a novel myocilin mutation in rural Philippines.

Abstract

Purpose : Juvenile onset primary open angle (JOAG) glaucoma is an important cause of blindness worldwide. We identified a novel disease-causing myocilin (MYOC) mutation in a large extended Filipino family residing in a remote Philippine island. MYOC genetic testing is necessary to identify individuals at risk for glaucoma in this remote region, however access to eye care and genetic services is difficult in this community. The purpose of this study is to develop a low-cost and easily accessible genetic testing platform for this community using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) based genetic diagnostic method, Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK). SHERLOCK can be performed in a clinic-based setting at reduced cost.

Methods : Using SHERLOCK, we developed an assay to detect a novel MYOC mutation (c.1515A>G .*505Wext*42) mutation using genomic DNA. CRISPR RNA guides were designed to detect the wildtype and mutant myocilin sequences. Isothermal recombinase polymerase amplification was used to amplify genomic DNA collected from affected and unaffected family members. A Cas13a based fluorometric molecular detection platform was utilized to detect this single base pair mutation. Detected sequence variants were confirmed using Sanger sequencing.

Results : The SHERLOCK platform detected the MYOCc.1515A>G mutation in 26 affected subjects tested. In addition the mutation was also detected in 7 children at risk for disease development. The overall cost of SHERLOCK was $2 per sample.

Conclusions : In the absence of a fully equipped diagnostic laboratory, the use of SHERLOCK is a sensitive, specific, field-deployable, and cost-effective method for detecting specific disease-causing mutations in communities with minimal health and diagnostic facilities. Our study has already identified 7 individuals at increased risk of glaucoma caused by a specific MYOC mutation, who will benefit from surveillance and early treatment. This approach gives hope for implementation of a population-based screening program in this remote island community and can serve as a template for future population-based genetic studies in other rural areas.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.