Sex as a biological variable. Our study examined a male patient; sex was not considered as a biological variable.

Cell culture and reagents. HEK293T and HEK293FT cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The human telomerase-immortalized RPE cells (hTERT-RPE1 or RPE1) were cultured in DMEM/F-12 (1:1) medium supplemented with 10% FBS and 1% penicillin-streptomycin. The human conjunctival fibroblasts (HConF, ScienCell Research Laboratories, 6570) from healthy human conjunctiva and conjunctiva cells from a patient with JBTS were cultured in DMEM/F-12 (1:1) medium supplemented with 10% FBS, Fibroblast Growth Supplement (FGS, ScienCell Research Laboratories, 2352), and 1% penicillin-streptomycin.

Plasmids. The human NDUFAF2, ARMC9, and TOGARAM1 cDNA was obtained from Origene (RC207387, RC209499, and RC222959). To generate the epitope-tagged ARMC9, fragments were amplified by PCR and cloned into pcDNA3-FH. The pcDNA3-FH vector was derived from pcDNA3 (Thermo Fisher Scientific) but contained sequences for FLAG and HA epitope tag between the HindIII and BamHI cloning sites. Thus, the expressing proteins expressed FLAG and HA epitope tag at the N-terminus. Various ARMC9 mutants were cloned to pcDNA3-FH vector and used to analyze ARMC9-NDUFAF2 interaction (Figure 4, A and B, and Supplemental Figure 3). The NDUFAF2 and ARMC9 fragments were also subcloned into pCS vector from Addgene (plasmid 12158) so that the constructs could be stably expressed in RPE1 cells, human conjunctival fibroblasts, and JBTS patient-derived fibroblasts.

Primary antibodies. Primary antibodies were obtained from the following sources and used according to the manufacturers’ instructions: rabbit anti-NDUFAF2 (Western blot [WB] 1:500; HPA054776, MilliporeSigma), mouse IgG2a anti-CEP164 (immunofluorescence [IF] 1:1,000; sc-515403, Santa Cruz Biotechnology), rabbit anti-CP110 (WB 1:1,000, IF 1:1,000; 12780-1-AP, Proteintech), mouse IgG2b anti-FOP (IF 1:1,000; H00011116-M01, Abnova), anti–polyglutamylated tubulin (pGlu-Tu) (IF 1:3,000; 901501, AdipoGen), rabbit anti–myosin-Va (IF 1:200; NBP1-92156, Novus Biologicals), rabbit anti-GAPDH (WB 1:5,000; 60004-1-Ig, Proteintech), mouse IgG2a anti-Arl13b (IF 1:500; 75-287, Antibodies Incorporated), rabbit anti-TOMM20 (IF 1:1,000; ab56783, Abcam), rabbit anti-ARMC9 (WB 1:500 in 5% BSA; HPA026671, MilliporeSigma), mouse IgG2b anti–8-oxo-Dg (IF 1:250; 4354-MC-050, R&D Systems), mouse anti-FLAG (WB 1:5,000; F3165, MilliporeSigma), rabbit anti-NPHP1 (IF 1:1,000; SAB1401267, MilliporeSigma), rabbit anti-Myc (WB 1:1,000; 2278, Cell Signaling Technology), mouse IgG1 anti–α-tubulin (WB 1:1,000; T6199, MilliporeSigma), mouse IgG1 anti-ZN5 (IF 1:1,000; ZDB-ATB-081002-19, Zebrafish International Resource Center, Eugene, Oregon, USA), mouse IgG1 anti-rhodopsin (IF 1:500; ab5417, Abcam), rabbit anti-recoverin (IF 1:500; AB5585-I, MilliporeSigma), rabbit anti-GAPDH (WB 1:1,000; 2118S, Cell Signaling Technology), rabbit anti-IFT88 (IF 1:1,000; 13967-1-AP, Proteintech), rabbit anti-CEP97 (WB 1:1,000, IF 1:1,000; 22050-1-AP, Proteintech), rabbit anti-CEP290 (WB 1:1,000, IF 1:1,000; A301-659A, Thermo Fisher Scientific), rabbit anti-MKS3 (IF 1:250; 13975-1-AP, Proteintech), rabbit anti-TCTN2 (IF 1:250; 17053-1-AP, Proteintech), rabbit anti-MKS1 (IF 1:200; 16206-1-AP, Proteintech), rat anti-ARMC9 (IF 1:200; 90703, BiCell Scientific), mouse IgG1 anti-Zrp1 (IF 1:1,000; ZDB-ATB-081002-43, Zebrafish International Resource Center), MitoTracker Red CMXRos (M7512, Thermo Fisher Scientific).

Transient transfection. Transient transfections were performed using TurboFect Transfection Reagent (Thermo Fisher Scientific, R0533). Three million 293T cells were plated on 60 mm culture dishes overnight. Cells were transfected with 2.5 μg of expression constructs according to the manufacturer’s instructions. Cells were harvested 48 hours after transfection.

Lentivirus production and infection. Five hundred thousand 293FT cells were plated on 60 mm dishes using TurboFect Transfection Reagent with the following plasmids: 1.5 μg of V-SVG, 2.5 μg pCMV-gag-pol, and 3.5 μg of the pCS-based constructs. The supernatant containing viral particles was harvested 48 hours after transfection. Virus-containing medium was passed through a 0.45 μm filter (Fisher Scientific, 13-100-105). For the infection of RPE1 cells, human conjunctival fibroblasts, and JBTS conjunctiva cells, 5 × 105 cells were seeded onto a 60 mm plate the night before infection and incubated with 3 mL of viral stock. The medium was changed to fresh culture medium 24 hours after infection. From 2 days after infection, cells were maintained in culture medium.

Generation of NDUFAF2- and ARMC9-knockout RPE1 cells. The targeting sequences for NDUFAF2 and ARMC9 were at exon 1 of NDUFAF2 (5′-GCAGTACAAGAACTGGAGAG-3′) and exon 3 of ARMC9 (5′-TTTCAAGTTCCATCCGAGAT-3′). The target sequences were cloned into the gRNA cloning vector (pSLQ1654) containing Cas9 via the restriction enzyme BbsI (Thermo Fisher Scientific, FD1014). Knockout cells were all obtained through clonal propagation from a single cell. For genotyping, the following PCR primers were used: 5′-TCCAGGATGGAGGCCGACCT-3′ and 5′-TAGTAGGGACCGCGGACGCA-3′ for NDUFAF2 alleles, and 5′-ATGGGGGACATTCTGGCTCAT-3′ and 5′-GAGGGGTGCACCATAGGGTTG-3′ for ARMC9 alleles. PCR products were cloned and sequenced.

Immunostaining. Cells were grown on 0.1 mg/mL of poly-l-lysine–coated coverslips and fixed with methanol at −20°C for 15 minutes. Cells were then washed 3 times with PBS and incubated in blocking buffer that contained 3% BSA (wt/vol) and 0.1% Triton X-100 in PBS for 30 minutes at room temperature (RT). Primary antibodies were all diluted in the blocking buffer and incubated for 2 hours at RT. Alexa Fluor 488–, 594–, or 647–conjugated goat secondary antibodies were used at 1:500 dilution (Thermo Fisher Scientific) and incubated for 1 hour at RT. DNA was visualized using DAPI (Thermo Fisher Scientific). Coverslips were mounted on the slides with mounting medium (ProLong Gold Antifade, Thermo Fisher Scientific). Fluorescent images were obtained using an LSM880 Zeiss confocal microscope. Images were acquired and processed by ZEN software (Carl Zeiss) or ImageJ software (NIH).

Immunoblotting. Cells were washed with ice-cold PBS twice and lysed in ice-cold RIPA lysis buffer (MilliporeSigma, 20-188) that contained protease inhibitor cocktail (Thermo Fisher Scientific, PI78430). Cell debris was removed by centrifugation at 13,500g for 15 minutes at 4°C. Protein concentrations were determined by the BCA Protein Assay (Thermo Fisher Scientific, 23227). Equal amounts of proteins were mixed with SDS sample buffer, boiled at 95°C for 5 minutes, and separated by SDS-PAGE. The resolved proteins were then transferred onto 0.2 μm nitrocellulose membranes (Bio-Rad, 1620097). Blots were blocked for 1 hour at RT with 5% nonfat milk in TBS-T (20 mM Tris, pH 7.6, 137 mM NaCl, and 0.1% Tween-20) and incubated overnight at 4°C with primary antibodies in blocking solution. Membranes were washed 3 times with TBS-T and incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies for 1 hour at RT (Invitrogen, 31430 and 31460). After washing 3 times with TBS-T, proteins were visualized with ECL Western blotting substrate (Thermo Fisher Scientific, 34095).

Immunoprecipitation. Transfected cells were lysed on 60 mm dishes in buffer that contained 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS together with phosphatase and protease inhibitor. One milligram of cell lysates were suspended in 1 mL lysis buffer and incubated with 5 μL of anti-FLAG M2 beads (MilliporeSigma) overnight at 4°C under gentle rotation. Beads were then washed 3 times with lysis buffer that contained protease inhibitors. The immunocomplex was eluted by SDS sample buffer and separated by gels for Western blot analysis.

Quantification of immunostaining images. ZEN software (Carl Zeiss) was used to quantify the fluorescent intensity of proteins at the centrioles and transition zone and to quantify cilia length. All cells were treated the same during immunostaining and image acquisition. The same setting was applied to all images. A circle was drawn around the centrioles and transition zone, and the total pixel value of the marked region was measured. The signal ratio of the marked region over the proteins at the centrioles was then normalized to the control group. All quantifications were obtained from at least 3 experiments.

Injection of morpholino antisense oligonucleotides into zebrafish embryos. Wild-type zebrafish embryos in the single-cell stage were washed and collected in E3 water (water containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) in a Petri dish. Two morpholinos (MOs) were purchased from Gene Tool Company: NDUFAF2-MO1, targeting the translation 5′-UTR of NDUFAF2 (5′-TTGTACTGCACATGCAAACACGTTC-3′), and NDUFAF2-MO2, targeting the translation start codon site of NDUFAF2 (5′-CAATGCGGCTCATCTCTGTGATTTA-3′). 0.4 mM morpholinos and 100 ng/μL RNA with a final concentration of 0.1% phenol red (MilliporeSigma, P0290) were injected into single-cell-stage embryos at the desired concentrations using a PLI-100A microinjector (Harvard Medical Apparatus). After microinjection, the embryos were placed in a Petri dish with E3 medium and incubated for development at 28°C.

In vitro microinjection of transcribed mRNA into zebrafish embryos. The cDNA of NDUFAF2 was cloned into a pcDNA3.1 vector, and large amounts of capped RNA were synthesized in vitro using the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen Ambion, AM1344M). RNA samples were then purified with the RNeasy Mini Kit (Qiagen, 74104). On the day of the injection, 100 ng/μL RNA samples were thawed, vortexed lightly, and spun down briefly with a final concentration of 0.1% phenol red (MilliporeSigma, P0290).

TEM imaging. HConF and JBTS cells were grown on Aclar film–based (Electron Microscopy Sciences) coverslips and fixed in 4% paraformaldehyde (Electron Microscopy Sciences, 15710) and 2.5% glutaraldehyde (MilliporeSigma, G5882) in PBS buffer at 37°C for 30 minutes. Cells were then further postfixed in 1% osmium tetroxide (OsO4) in PBS buffer for 30 minutes on ice. After dehydrating in a graded series of ethanol, the cells were then infiltrated and embedded in EPON812 resin (Electron Microscopy Sciences, catalog 14120) to generate a resin sample. A microtome (Ultracut UC6, Leica) was used to cut the sample into serial sections (~90 nm thickness), which were then stained with 1% uranyl acetate and 1% lead citrate. Samples were imaged using the JEOL JEM1400 transmission electron microscope with a LaB6 emitter.

Real-time PCR. Real-time PCR was carried out in an Applied Biosystems Inc. StepOne Real-Time PCR System using the Maxima SYBR Green/ROX qPCR Master Mix (2×) (Thermo Fisher Scientific, K0221). Primers for 1 rhodopsin marker, 2 transducin markers (gnat1 and gnat2), and 1 nuclear DNA marker (β-actin) were used. Amplifications were performed in 20 μL reaction mixtures consisting of 100 ng total DNA, 1× SYBR Green PCR Master Mix with 0.5 μM of each primer. Triplicate reactions were performed for each marker in a 96-well plate using a 3-step amplification program of initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The comparative cycle threshold (Ct) method was used to analyze the data by generating relative values of the amount of rhodopsin and transducin. Relative content was calculated after determination of the difference between Ct of the given rhodopsin marker and 2 transducin markers and that of the calibrator nuclear marker β-actin. Each measurement was repeated in triplicate, and a non-template control was included in each experiment. The sequences of each gene were: rhodopsin (zebrafish), forward, 5′-AGTCCTGCCCAGACATCTAG-3′; rhodopsin, reverse, 5′-GTACTGTGGGTATTCGTATGGG-3′; gnat1, forward, 5′-CGTCAAGTTTGTGTTCGATGC-3′; gnat1, reverse, 5′-GAGGAAACGAGCTACAAGGAG-3′; gnat2, forward, 5′-CAAACCTGACTACCTTCCCAC-3′; gnat2, reverse, 5′-TCTTCCTCTCGGACCTCTG-3′; β-actin, forward, 5′-TGCTGTTTTCCCCTCCATTG-3′; and β-actin, reverse, 5′-GTCCCATGCCAACCATCACT-3′.

Oxygen consumption rate. Oxygen consumption rate (OCR) was measured using a Seahorse Biosciences XFe96 extracellular flux analyzer. 1.25 × 105 cells per well were seeded in XFe96 cell culture plates. Attachment of the cells was monitored after 24 hours, and cells were incubated overnight at 37°C with 5% CO2. Before the assay, cells were changed to Seahorse XF DMEM medium with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose and equilibrated for 1 hour at 37°C without CO2. The OCR was measured afterward using the following inhibitors: 2.5 μM oligomycin, 2 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and 0.5 μM rotenone with 0.5 μM antimycin A (Agilent Technologies, 103015-100). For each condition, cycles were performed in triplicate with 3 minutes of mixing followed by 3 minutes of measurement. After completion of the assay, the cell number per well was determined using the Cytation 5 (Agilent Technologies). The OCR was normalized to the cell number for each well.

Catalytic activity of mitochondrial complex I. The procedure followed the previously published protocol (74). The fibroblast lysate was suspended in 1 mL of 10 mM ice-cold hypotonic Tris buffer (pH 7.6). The suspension was then taken up and expelled several times using a 26 gauge needle until it became a homogeneous solution. After the addition of 200 μL of 1.5 M sucrose, the homogeneous solution was centrifuged at 600g for 10 minutes at 4°C. To collect the supernatant, the solution was centrifuged at 14,000g for 10 minutes at 4°C, and the mitochondrial pellet was then suspended in 0.5 mL of 10 mM ice-cold hypotonic Tris buffer (pH 7.6). The homogeneous solution was frozen and thawed in liquid nitrogen and at 37°C three times. Enriched mitochondrial fractions were purified from fibroblast lysates. Complex I activity in cells was analyzed by determining rotenone-sensitive activities followed by detection of 340 nm wavelength.

Assay of NAD+/NADH ratio. The procedure followed the protocol of the Abcam NAD/NADH Assay Kit (ab65348). Two million cells were washed in 1× PBS and lysed in 500 μL buffer (0.25 M sucrose, 10 mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor). Cells were placed on ice for 15 minutes and sheared with a 26 gauge needle, and homogenates were centrifuged at 700g for 5 minutes at 4°C. Supernatants were then collected and centrifuged at 10,000g for 30 minutes at 4°C. The pellet was the mitochondrial fraction. The pellet was resuspended in 400 μL NADH/NAD extraction buffer, and cells were extracted by 2 freeze/thaw cycles (20 minutes on dry ice followed by 10 minutes at RT). Extracted cells were vortexed for 10 seconds and then centrifuged for 5 minutes at 4°C at top speed in a cold microcentrifuge to remove any insoluble material. The supernatant was separated, and enzyme was removed by passage through a 10kD Spin Column (Abcam, ab93349) before performance of the assay. For the NADH assay, half of the extraction sample was aliquoted and heated at 60°C for 30 minutes. The standard curve was prepared, and samples loaded into 96-well plates according to the manufacturer’s instructions (Abcam, ab65348).

Image analysis of mitochondrial length. Mitochondrial dynamics were analyzed from fluorescence microscope images using Mytoe, software developed by Andre Ribeiro’s laboratory (75).

Proximity ligation assay. Five hundred thousand cells were seeded on cover slides and fixed with 4% paraformaldehyde in PBS for 20 minutes at RT. For ARMC9 antibody, the initial fixation was followed by fixation in ice-cold 100% methanol for 15 minutes at –20°C. The slides were then washed twice with PBS, and cells were blocked with Duolink block solution (MilliporeSigma, DUO92101-1KT) for 1 hour at RT. During proximity ligation assays (PLAs) (MilliporeSigma, DUO92101), the primary antibodies were diluted in Duolink antibody dilution buffer (MilliporeSigma, DUO92101-1KT) and stained overnight at 4°C. The next day, the slides were washed with a large volume of 5% BSA in PBS for 10 minutes. Before the PLA assay, a 20 μL total volume per reaction of secondary antibodies was prepared with 4 μL Anti–rabbit PLUS, Affinity-purified Donkey Anti–rabbit IgG (H+L) antibody and 4 μL Anti–mouse MINUS, Affinity-purified Donkey Anti–mouse IgG (H+L) antibody plus 12 μL Duolink antibody dilution buffer for 20 minutes at RT. Secondary antibodies were then added onto slides and incubated at 37°C for 1 hour. Before the ligation process of the PLA assay, the slides were washed with 1× of buffer A twice for 5 minutes. During the ligation process, the prepared 20 μL total volume per reaction of ligation mix was added onto slides with 4 μL ligation stock, 15.5 μL double-distilled H2O (ddH2O), and 0.5 μL ligase for 30 minutes at 37°C. Before the amplification process of the PLA assay, the slides were washed by 1× of buffer A twice for 5 minutes. During the amplification process, the 20 μL total volume per reaction of amplification mix was prepared and added onto slides with 4 μL amplification stock, 15.75 μL ddH2O, and 0.25 μL polymerase for 100 minutes at 37°C. After that, the slides were washed twice with 1× of buffer B for 10 minutes and once with 0.01× of buffer B for 1 minute. After aspiration of buffer B, cells were mounted in ProLong Gold Antifade Reagent with DAPI (MilliporeSigma, DUO82040).

Fluo-4 AM. The Fluo-4 AM, cell permeant (F14201, Invitrogen), stock solution was prepared in DMSO at a concentration of 1 mM before loading of the cells with the product. A final concentration of 2 μM in E3 buffer was achieved followed by incubation at 28°C for 15 minutes. Ca2+ binding was used to indicate fluorescent signal (excitation/emission peaks at 494/506 nm), with detection in the time-lapse live images under a confocal microscope.

Study subjects, skin biopsy, and fibroblast culture. Family 1 was identified at Indiana University, and family 2 was identified at Lucile Packard Children’s Hospital Stanford. A fibroblast sample was harvested from surgical waste (removed conjunctiva) from the proband in family 2. The biopsy was minced, transferred to the bottom of a T-25 culture flask, and cultured in minimum essential medium supplemented with 10% FBS, 1% non-essential amino acids, and 1% antibiotics/antimycotics. Cultures were maintained at 37°C in a 5% CO2 and 5% O2 incubator, and medium was replaced after 1 week. Once the fibroblasts had grown to approximately 50% confluence, cells were trypsinized and replated into a T-75 flask. Cells were then collected and frozen at –80°C.

Functional measurements of ocular motility. Eye movements were quantified using Tobii Pro Spectrum (Tobii Technology AB) infrared video oculography. This system uses dual near-infrared cameras to sample the position of the subject’s eyes at a rate of 1,200 Hz with an accuracy of 0.3°. The subject was positioned 61 cm from the screen, without a chin rest. A child-appropriate visual stimulus pattern was used with the Tobii Pro Lab software on a 24-inch monitor (resolution 1,920 × 1,080 pixels). A stimulus target of a small cartoon animal spanning 128 by 165 pixels was placed in the center of the monitor to induce fixation and then moved to each of 4 eccentric positions (252 pixels in 4 cardinal positions from center fixation) to stimulate reflexive saccades. After the eye tracking recording, fixation and saccade data were analyzed using Tobii Pro Lab software and oculometrics quantified in 5 areas of interest (AOIs; Tobii Pro Lab), including gaze duration, fixation count, saccade count, and scan pattern.

Statistics. All data are presented as mean with standard deviation (SD) from at least 3 independent experiments (GraphPad Prism 8). Experimental samples and numbers for statistical testing are reported in the corresponding figure legends. To evaluate the differences among 3 or more groups, 1-way ANOVA was used, followed by a Tukey-Kramer multiple-comparison test. For 2-group comparisons, a 1-tailed Student’s t test was used for normally distributed data. P values less than 0.05 were considered to indicate statistical significance.

Study approval. The families consented to studies approved by the Indiana University Institutional Review Board (IRB 3023) and Stanford University Institutional Review Board (IRB-32223) and gave written permission to publish photographs (where applicable) and the pertinent clinical information. All study participants were informed of the purpose of the examination, and all the participating individuals signed a written informed consent prior to the study, ensuring their voluntary participation. The overall study was approved by the Institutional Review Board of Stanford University for studies involving humans (IRB-5537 and IRB-39756) and adhered to the tenets of the Declaration of Helsinki and ethical guidelines and protocols to protect the participants’ privacy and well-being.

Data availability. All the data sets generated for this study can be found within the article figures and supplemental material. Values for all data points in graphs are reported in the Supporting Data Values file.