SUMMARY Neuronal arborization is regulated by cell autonomous and non-autonomous mechanisms including endosomal signaling via BDNF/TrkB. The endosomal Na+/H+ exchanger 6 (NHE6) is mutated in a new autism-related disorder. NHE6 functions to permit proton leak from endosomes yet the mechanisms causing disease are unknown. We demonstrate that loss of NHE6 results in over-acidification of the endosomal compartment and attenuated TrkB signaling. Mouse brains with disrupted NHE6 display reduced axonal and dendritic branching, reduced synapse number and circuit strength. Site-directed mutagenesis shows that the proton leak function of NHE6 is required for neuronal arborization. We find that TrkB receptor co-localizes to NHE6-associated endosomes. TrkB protein and phosphorylation are reduced in NHE6 mutant neurons in response to BDNF signaling. Finally, exogenous BDNF rescues defects in neuronal arborization. We propose that NHE6 mutation leads to circuit defects that are in part due to impoverished neuronal arborization that may be treatable by enhanced TrkB signaling.

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 MEASURESA 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.

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Endoscopic vitrectomy is a useful and unique adjunct to microincision vitreoretinal surgery. The optical properties of endoscopy allow for some clinically advantageous approaches that are not possible with regular microscope viewing systems, namely, the ability to both bypass optically signficant anterior segment opacities and directly visualize dificult-to-access retroirideal, retrolental, and anterior retinal structures in their natural anatomical configuration. The surgical benefits include improved surgical access to the pars plana, pars plicata, ciliary sulcus, ciliary body, and peripheral lens, along with unique access to anterior traction in complex pediatric anterior detachments, particularly in retinopathy of prematurity. This review will focus on the development and surgical utility of intraocular endoscopy, provide an update on its current uses in the era of microincision vitreoretinal surgery, and highligh its role in pediatric vitreoretinal diseases.

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Purpose: To report on the indications, outcomes, and complications of endoscopic vitrectomy in a large cohort of pediatric vitreoretinal patients. Methods: This is a retrospective interventional case series consisting of 244 eyes of 211 patients aged 18 years or younger undergoing a total of 326 endoscopic vitrectomies from 2008 to 2017. A 23-gauge vitrectomy was performed with use of a 19-gauge endoscope. Results: Two hundred and eleven patients with a mean age of 7.5 years (range: 0–18 years) and median follow-up since last surgery of 28 months (range: 3 months–8.7 years) were included. The most common indication for endoscopic vitrectomy was retinal detachment (234/326; 72%) with proliferative vitreoretinopathy (162/234; 69%). Other diagnoses included trauma (25%), retinopathy of prematurity (15%), and glaucoma (9%). Twenty-five percent of surgeries (80/326) were performed on eyes with significant corneal opacities. Retinal reattachment was achieved in 67% of eyes with retinal detachment (119/178). Visual acuity improved in 26% of retinal detachment eyes versus 53% of nonretinal detachment eyes (P = 0.005). Surgical complications included band keratopathy (15%), hypotony (8%), cataract (7%), and elevated intraocular pressure (3%). Conclusion: In this large series of pediatric endoscopic vitreoretinal surgeries, anatomic outcomes and complication rates were comparable with previous studies.

Chronic granulomatous disease (CGD) is a rare, genetically heterogeneous condition that occur from mutations in NADPH oxidase, resulting in recurrent infections from catalase-positive organisms and granuloma formation. It is most commonly diagnosed within the first two years of life. We present a case of a six-month-old infant who presented with left tibial pain with initial concerns for child abuse and was found to have CGD. A skeletal survey revealed multiple lytic lesions, diffuse osteopenia, and opacities in the lungs. MRI revealed a tibial subperiosteal abscess and, after incision and drainage, cultures grew Serratia marcescens, an unusual catalase-positive organism. Targeted testing for immunodeficiency revealed 0% NADPH oxidase activity on nitro blue tetrazolium (NBT), confirming the diagnosis.