Department of Pediatrics, Children's Hospital Los Angeles, Los Angeles, California.
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, Bethesda, Maryland.
JAMA Ophthalmol. 2019 Dec 1;137(12):1381-1388. doi: 10.1001/jamaophthalmol.2019.3914.
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.
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.
A functional assay was used to confirm the pathogenicity of novel RPE65 VUSs in 4 patients with Leber congenital amaurosis.
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.
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.
下一代测序可以检测到意义不明的变异（VUS），对于其中一些，基因治疗并没有优势。因此，在进行双侧玻璃体切除术和施用 voretigene neparvovec-rzyl 之前，应确认复合杂合子或纯合子变异的致病性。
描述一种用于评估 RPE65 基因变异致病性的体外诱变检测。
设计、设置和参与者：这是一项在 2 个三级转诊中心进行的病例系列研究。回顾了 2008 年 9 月 5 日至 2019 年 12 月 31 日的临床病史、影像学和电生理检查结果。参与者为 4 名患有莱伯先天性黑矇的儿科患者，他们接受了 1 期至 3 期临床试验的评估或符合纳入标准，或因接受 voretigene neparvovec-rzyl 治疗而被转介。
使用功能检测法确认了 4 名莱伯先天性黑矇患者的 RPE65 新 VUS 的致病性。
4 名莱伯先天性黑矇患者的 RPE65 存在 VUS。患者 1 和 2 是同胞，携带纯合子 VUS c.311G>T p.(G104V)。患者 3 是复合杂合子，有 1 个已知的致病性等位基因 c.1202_1203insCTGG p.(Glu404AlafsTer4)和 1 个 VUS c.311G>T p.(G104V)，这两个变异分别位于不同的等位基因上。患者 4 也是复合杂合子，有 1 个致病性变异 c.11+5G>A 和 1 个位于反式的变异 c.1399C>T p.(P467S)。体外诱变显示，G104V 和 P467S RPE65 蛋白无催化活性（仅有 0%异构酶活性）。由于腺相关病毒 2 衣壳中和抗体滴度较高，患者 1 和 2 被排除在 1 期临床试验之外。患者 3（G104V）和 4（P467S）接受了 voretigene neparvovec-rzyl 的成功手术基因治疗，他们对低白光强度和视野的反应在基因治疗干预后不到 30 天内增加。
本研究结果表明，在 RPE65 错义突变患者中，可对蛋白功能进行功能检测，以评估复合杂合子和纯合子病例中变异的致病性。鉴于基因治疗手术的潜在风险，应考虑进行体外 RPE65 活性检测，以避免治疗假基因型的可能性。
