Figure 1.

Compound heterozygous mutations in the UGDH gene caused decreased enzymatic activity, interruption of the cell cycle, small brain and neuronal loss in zebrafish F0 crispant. (A) The pedigree of family 1 segregates CM. The arrow points to the proband. Compound heterozygous mutations c.71C > T and c.404G > A in UGDH are presented below individuals. (B) The pedigree of the family 2 segregates CM. The arrow points to the proband.Compound heterozygous mutations c.169C > A and c.731C > A in UGDH are presented below individuals. (C, D) Purified UGDH wild-type (WT) and mutant enzymatic activity. (C) A24V and R135Q in patient 1 and (D) L57I and T244K in patient 2 were measured as the turnover of NAD⁺ to NADH. The assay was performed by at least three replicate experiments. (E) Percentage of cells in different phases of the cell cycle (G0/G1, S, G2/M). KD, UGDH knockdown HEK293 cell. Ctrl, controls. ^∗∗P < 0.01, ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001; student's t-test. ns indicates no significance with a P-value >0.05. (F) Representative bright-field imaging of larval zebrafish at 5 days after fertilization (dorsal view). Top, cas9 injected control; bottom, ugdh F0 CRISPR (crispant). The Red plus blue line indicates the body length and the red line indicates the head length. (G) Representative imaging of HuC: eGFP-expressed larval zebrafish shows CNS fluorescence pattern at 5 after fertilization (dorsal view). Left, cas9 injected control; right, ugdh crispant. (H–J) Measurements of body length, head length, and the head-to-body length ratio in cas9 injected control (n = 20 fish) versus ugdh crispant (n = 28 fish). Body length and head length data were normalized to the mean value of cas9 control group. (K) Normalized CNS fluorescence area in cas9 injected control (n = 20 fish) versus ugdh crispant (n = 28 fish). Scale bars are indicated in the figure. Error bars indicate standard deviation (SD). Statistical significance is indicated as ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.