Identification and phenotypic expression of RDH12-related retinitis pigmentosa by panel-based NGS and CRISPR/Cas9 gene editing

Abstract

Purpose : Retinitis pigmentosa (RP) is an inherited retinal disease (IRD) with heterogeneous genetic background. It leads to progressive photoreceptor degeneration and severe visual disturbance. We performed panel-based next generation sequencing (NGS) for genetic diagnosis of patients, and the variants of interest could be introduced to zebrafish by using CRISPR/Cas9. Here, we reported the phenotypic expression of a RDH12 variant in zebrafish for validation of diagnosis.

Methods : The reported case was recruited in the TIP (Taiwan Inherited retinal diseases Project) program. The blood sample was collected from a 43-year old woman with RP symptoms. The genomic DNA was sequenced by panel-based NGS with 215 genes associated with IRDs. NGS data were processed and pathogenicity of retained variants was predicted by SIFT (score < 0.05) and PolyPhen-2 (score > 0.99) algorithm. The criteria of classifying pathogenic variants were based on the ACMG standard and guideline. The selected variants were introduced to zebrafish by CRISPR/Cas9. Retinal morphology and immunochemistry were then studied.

Results : There are more than 300 probands recruited in the TIP program and we performed in silico analysis of variants. In this case, the variant c.806C>G of RDH12 was identified to verify the phenotypic expression of variant in zebrafish by CRISPR/Cas9. Sequencing alignment of the proteins encoded by the zebrafish and human RDH12 orthologs was performed, and the dsDNA that contains variant and fluorescent reporter system (IRES-hrGFP) as a donor was introduced into targeted genome by delivering a guide RNA and a CRISPR-associated endonuclease Cas9. DNA sequencing was implied to verify the mutation in zebrafish after gene editing. Serial observations revealed obvious rod and cone degeneration and strengthened the results of genetic diagnosis.

Conclusions : Mutation c.806C>G in RDH12 gene was reported in late-onset cone-rod dystrophy before but not in RP patient while c.806_810delCCCTG could cause RP-like symptoms. Here, we report a case of compound heterozygous c.806C>G (p.A269G) transition in RDH12 who exhibits RP symptoms. With CRISPR/Cas9, a powerful gene editing method, and zebrafish animal model, we can further validate the variant by in vivo analysis. In conclusions, the variants that are selected form NGS data is introduced to zebrafish, and phenotypic expression of variants is confirmed.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.