Utility of In Vitro Mutagenesis of RPE65 Protein for Verification of Mutational Pathogenicity Before Gene Therapy

Question Can the pathogenicity of RPE65 variants of uncertain significance be identified before gene therapy? Findings In this case series of 4 pediatric patients with Leber congenital amaurosis, compound heterozygous or homozygous variants of uncertain significance were found in the RPE65 gene. An in vitro assay incorporated these mutations into RPE65 and showed catalytic inactivity, which established eligibility for treatment. Meaning This study suggests that in vitro testing of RPE65 protein function can be used to confirm the pathogenicity of compound heterozygous or homozygous variants of uncertain significance in the RPE65 gene to identify patients eligible for gene therapy surgical intervention. Importance Next-generation sequencing can detect variants of uncertain significance (VUSs), for some of which gene therapy would not be advantageous. Therefore, the pathogenicity of compound heterozygous or homozygous variants should be confirmed before bilateral vitrectomy and administration of voretigene neparvovec-rzyl. Objective To describe an in vitro mutagenesis assay for assessing the pathogenicity of variants in the RPE65 gene. Design, Setting, and Participants This case series was conducted at 2 tertiary referral centers. Clinical history, imaging, and electrophysiologic testing results were reviewed from September 5, 2008, to December 31, 2019. Participants were 4 pediatric patients with Leber congenital amaurosis who were evaluated for or met the inclusion criteria for phase 1 to 3 clinical trials or were referred for voretigene neparvovec-rzyl treatment. Main Outcomes and Measures A functional assay was used to confirm the pathogenicity of novel RPE65 VUSs in 4 patients with Leber congenital amaurosis. Results Four patients with Leber congenital amaurosis had VUSs in RPE65. Patients 1 and 2 were siblings with the homozygous VUS c.311G>T p.(G104V). Patient 3 was a compound heterozygote with 1 known pathogenic allele, c.1202_1203insCTGG p.(Glu404AlafsTer4), and 1 VUS, c.311G>T p.(G104V), which segregated to separate alleles. Patient 4 was also a compound heterozygote with 1 pathogenic variant, c.11 + 5G>A, and 1 variant in trans, c.1399C>T p.(P467S). In vitro mutagenesis revealed that the G104V and P467S RPE65 proteins were catalytically inactive (0% isomerase activity). Patients 1 and 2 were excluded from participation in a phase 1 trial owing to high Adeno-associated virus 2 capsid-neutralizing antibodies. Patients 3 (G104V) and 4 (P467S) underwent successful surgical gene therapy with voretigene neparvovec-rzyl, and their response to lower white light intensity and visual field increased in fewer than 30 days after gene therapy intervention. Conclusions and Relevance Findings from this study suggest that, in patients with missense mutations in RPE65, functional assays of protein function can be performed to assess the pathogenicity of variants in both compound heterozygous and homozygous cases. Given the potential risks of gene therapy operations, in vitro RPE65 activity testing should be considered to avoid the possibility of treating a false genotype. This case series describes an in vitro mutagenesis assay performed to confirm pathogenicity in 4 children or adolescents with Leber congenital amaurosis.