Identification of OGDHL variants in 12 families and mapping of known variants. A Pedigrees and segregation data for the 12 families included in this study. Affected and unaffected individuals are indicated by filled and open squares (males) and circles (females), respectively, and a triangle represents a pregnancy. Affected individuals are indicated by black arrows. Double lines indicate consanguinity. Genetic diagnoses were made in 14 individuals. B The position of changed coding sequence in genomic DNA and the drawing of resulting variants. Seven previously uncharacterized variants described in this study are marked in bold blue. C Seven out of eight missense variants from our studies are highly conserved across species

Neuroimaging findings of individuals with OGDHL pathogenic variants. Brain MRI findings of individual 1 (A-D), 6 (E–H), and 7 (I-L). Sagittal T2 (A and I) and T1 (E) weighted images showed markedly hypoplastic corpus callosum in individual 1 (A) and 7 (I) and dysplastic corpus callosum in individual 6 (E) with hypoplastic rostrum, genu, and anterior body and absent posterior body and splenium (yellow arrows). Mega cisterna magna was present in all affected individuals (A, E, and I, blue arrows), and individual 6 also had inferior vermian hypoplasia and widening of the foramen of Magendie (E, asterisk). Axial T2-weighted images (B, C, F, G, J, and K) revealed varying degrees of diffuse white matter volume loss, most severe in individual 6 (F and G) and 7 (J and K). Individual 7 also had scattered areas of leukomalacia (J, red arrows) and prominent involvement of brainstem and cerebellar white matter. Individual 6 had ventriculomegaly and colpocephaly (F, orange arrows). Coronal T2 (D and L) and T1 (H) weighted images showed hypoplastic olfactory bulbs in individual 1 (D) and 7 (L) (green arrows) and hypoplastic hippocampi in individual 6 (H, purple arrows)

The morphological phenotypes of zebrafish ogdhl F₀ knockouts. A Representative embryo images of uninjected, ogdhl F₀ and ogdhl F₀ co-injected with 200 picogram (pg) human OGDHL mRNA (F₀ + OGDHL) at 3 dpf. Arrow denotes heart edema. B,C Size measurements for head (blue line) and eye (red line) as indicated from genotypes in A were calculated as a percentage difference compared to the mean value of the uninjected embryos. D The presence of heart edema (black arrow in A) was calculated as a percentage of total embryos of each group positive for edema. E Locomotor activities of zebrafish larvae in light and dark conditions at 5 dpf, n = 96 larvae for each group. The larvae were habituated in the dark for 30 min, followed by three cycles of 10-min periods of light and dark. Error bars represent the mean ± SEM. Dark period (D), light period (L). F Average cumulative distance traveled by each larva during three cycles of either light or dark periods. Error bars = mean ± SD. For B, C, and F, each dot represents one embryo, and the mean value of each group is indicated at the bottom of the respective bar in the figure. For B–D, the number of embryos for uninjected = 57, Cas9 protein-injected (Cas9 inj.) = 60, ogdhl F₀ = 63, F₀ + OGDHL (100 pg) = 34, F₀ + OGDHL (150 pg) = 35 and F₀ + OGDHL (200 pg) = 32. Error bars = mean ± SD. Statistical significance was calculated by Brown–Forsthye and Welch’s ANOVA with Dunnett’s T3 multiple comparisons test: not significant (ns) p ≥ 0.05, *p < 0.05, ***p < 0.001, and ****p < 0.0001

Comparing mRNA rescue levels of OGDHL and OGDH in Dhtkd1 and Ogdh paralog-deficient zebrafish. A Representative image of uninjected, Cas9-injected, single knockout (dhtkd1, ogdha, and ogdhb F₀), double knockout (ogdha;ogdhb F₀), and triple knockout (ogdha;ogdhb;ogdhl F₀) embryos at 3 dpf. B, C Size measurements for head and eye with or without human OGDHL co-injection. The number of embryos for uninjected = 66, Cas9-injected = 60, dhtkd1 F₀ = 36, dhtkd1 F₀ + OGDHL = 36, ogdha F₀ = 66, ogdha F₀ + OGDHL = 36, ogdhb F₀ = 66, ogdhb F₀ + OGDHL = 36, ogdha;ogdhb F₀ = 36, ogdha;ogdhb F₀ + OGDHL = 36, ogdha;ogdhb;ogdhl F₀ = 36 and ogdha;ogdhb;ogdhl F₀ + OGDHL = 36. D, E The size measurements for head and eye with or without human OGDH co-injection. The number of embryos for uninjected = 60, dhtkd1 F₀ = 36, dhtkd1 F₀ + OGDH = 36, ogdha F₀ = 36, ogdha F₀ + OGDH = 36, ogdhb F₀ = 36, ogdhb F₀ + OGDH = 36, ogdhl F₀ = 36, and ogdhl F₀ + OGDH = 36. All values were calculated as a percentage difference compared to the mean value of the uninjected embryos. Each dot represents one embryo, and the mean value of each group is indicated at the bottom of the respective bar in the figure. Error bars = mean ± SD. Statistical significance was calculated by Brown–Forsthye and Welch’s ANOVA with Dunnett’s T3 multiple comparisons test: not significant (ns) p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Cell apoptosis was activated by ogdhl loss-of-function. A–C Representative image of uninjected, ogdhl F₀ and ogdhl F₀ co-injected with human OGDHL embryos at 3 dpf after TUNEL staining. Scale bars = 100 μm. A Lateral view of eyes. Anterior to the left and dorsal to the top. B Dorsal view of brains. Anterior to the left. C Lateral view of trunks. Anterior to the left and dorsal to the top. D–G Quantification of the number of TUNEL-positive cells in the eye, midbrain, hindbrain, and spinal cords. The mean value of each group is indicated at the bottom of the respective bar in the figure. TUNEL-positive cells were calculated as a percentage difference compared to the mean value of uninjected embryos. In D, each dot represents one eye. uninjected = 31, ogdhl F₀ = 36 and F₀ + OGDHL = 38 eyes. In E–G, each dot represents one animal. uninjected = 16, ogdhl F₀ = 18 and F₀ + OGDHL = 19 animals. Error bars = mean ± SD. Statistical significance was calculated by Brown–Forsthye and Welch’s ANOVA with Dunnett’s T3 multiple comparisons test: not significant (ns) p ≥ 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Zebrafish ogdhl F₀ mutant displays motor neuron abnormalities. A–L Representative image of the trunk region of uninjected, Cas9-injected, ogdhl F₀ and F₀ + OGDHL in Tg(olig2:DsRed2;mnx1:GFP);Albino embryos at 3 dpf. M The measurement of axon length was started from spinal cord to the end of latest posterior branch (indicated by red arrow) of caudal primary motor neurons (CaP) (indicated by red line). Axon lengths were calculated as a percentage difference compared to the mean value of uninjected embryos. Each dot represents one axon. Uninjected = 24, Cas9-injected = 24, ogdhl F₀ = 30, and F₀ + OGDHL = 26. N The measurements of axon angle (indicated by magenta lines, start from spinal cord). Each dot represents one axon. n = 30 axons for each group. Error bars = mean ± SD. The mean value of each group was indicated at the bottom of the respective bar in the figure. Statistical significance in M and N was calculated by Brown–Forsthye and Welch’s ANOVA with Dunnett’s T3 multiple comparisons test: not significant (ns) p ≥ 0.05, **p < 0.01, and ****p < 0.0001. O Cartoon figures indicate aberrant motor axons observed in ogdhl F₀ embryos such as cross somite segment (blue hash) or missing EGFP signal (magenta asterisk). P Quantification of aberrant motor axon as indicated in O. n = 30 axons for each group

Functional characterization of human OGDHL variants in zebrafish model. A Experimental approach for characterizing functionality of human OGDHL variants. Human OGDHL variant mRNAs were mixed with synthetic zebrafish-specific ogdhl single-guide RNAs (sgRNAs) and Cas9 protein and microinjected into one-cell stage embryos, followed by phenotypic evaluation at 3 dpf. Blue line indicates head size measurement approach. Red line indicates eye size measurement. Green arrowhead indicates heart edema. B, C Head and eye size measurements were calculated as a percentage difference compared to the mean value of the uninjected embryos. Subsequently, the calculated measurements of uninjected, Cas9-injected, and ogdhl F₀ embryos were compared. Additionally, the ogdhl F₀ co-injected with either WT or mutated OGDHL mRNA were compared to the ogdhl F₀ knockout embryos. Each dot represents one animal and the mean value of each group is indicated at the bottom of the respective bar in the figure. n = the number of embryos. Error bars = mean ± SD. Statistical significance was calculated by Brown–Forsthye and Welch’s ANOVA with Dunnett’s T3 multiple comparisons test: not significant (ns) p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Significances for all rescue experiments are in comparison to ogdhl F₀. D The presence of the heart edema phenotype was calculated as a percentage of total embryos of each group. E The schematic of OGDHL truncating variants and the corresponding rescue experiment results