Electron transport chain paired and individual complexes in muscle biopsy specimen are depicted, which shows decreased paired and individual complex activities in Patient 1 compared to controls.

Vitamin B6 vitamers levels are reduced in aldh7a1 homozygous knock-out embryos relative to the wildtype. It is shown in a scatterplot of quantification of the levels of vitamin B6 vitamers in aldh7a1 homozygous knock-out zebrafish relative to the wildtype as measured by LC-MS. The graph demonstrates decreased levels of vitamin B6 vitamers in aldh7a1 knock-out 11 dpf embryos (aldh7a1 knock-out, grey squares) relative to the age-matched wildtype control embryos (Wildtype, black circles). Each dot represents metabolite data collected from 20 embryos. For a single data point of homozygous aldh7a1 knock-out zebrafish, only 16 embryos were available, so that data point was excluded from the analysis. nd, not detectable–the levels of metabolites could not be measured. p values were calculated by unpaired student t-test for multiple measurements followed by Holm-Sadak correction for multiple analysis. Error bars, SD.

TCA cycle metabolite levels are reduced in aldh7a1 knock-out zebrafish embryos. Scatterplot of quantification of the TCA cycle metabolite levels decreased in aldh7a1 11 dpf knock-out embryos (aldh7a1 knock-out, grey squares) relative to the wildtype age-matched controls (Wildtype, black circles), as measured by LC-MS/MS. Each dot represents metabolite data collected from 20 embryos. p values were calculated by unpaired student t-test for multiple measurements followed by Holm-Sadak correction for multiple analysis. Error bars, SD.

Electron transport chain enzyme activities are reduced in aldh7a1 knock-out zebrafish. 5A: individual and paired complexes and citrate synthase. 5B: ratio of individual and paired complexes to citrate synthase. Bar graph of the quantification of fold change in the electron transport chain enzyme activities in knock-out aldh7a1 11 dpf embryos (grey bars) relative to the wildtype age-matched controls (black bars). Each bar represents data collected from 300 embryos. The activity of each enzyme was measured in presumed homozygous 300 and 300 wildtype embryos. The values are depicted as enzyme activity in nmol/min/ mitochondrial protein mass normalized to the activity of citrate synthase (For further details please refer to Materials and Methods).

qPCR graph of mtDNA amount from wildtype (Wildtype, black circles) and aldh7a1 homozygous knock-out (aldh7a1 knock-out, grey squares) zebrafish embryos, demonstrating only a slight reduction in mtDNA copy number in knock-out relative to the wildtype embryos. Each dot represents DNA extracted from 5 embryos. The amounts of mtDNA from knock-out and wildtype embryos were normalized to the average mtDNA amount from the wildtype embryos. p value is calculated by an unpaired two-tailed student t-test. Error bars, mean and SD.

The levels of GABA metabolism reaction intermediates are reduced in aldh7a1 knock-out zebrafish embryos. It is shown in a scatterplot of quantification of the levels of GABA metabolism pathway metabolites, as measured by GC-MS and LC-MS/MS. The metabolite levels in 11 dpf aldh7a1 homozygous knock-out (aldh7a1 knock-out, grey squares) embryos are significantly decreased relative to the wildtype (Wildtype, black circles). Each dot represents metabolite data collected from 20 embryos. p values were calculated by unpaired student t-test for multiple measurements followed by Holm-Sadak correction for multiple analysis. Error bars, SD.