Fig. 1

Clinical and genetic characterization. a Brain MRI of the patient at fetal 34 weeks, birth, 1 month, and 7 months, revealing evidence of cerebellar hypoplasia. b Muscle MRI in the right gluteus maximus muscle at 14 months. c Muscle biopsy from gastrocnemius at 14 months. The diameter of muscle fibers range between 5 and 50 μm, with occasional degenerated and regenerated muscle fibers. Scale bar: 50 μm d Sequence chromatograms of GMPPB gene of the patient. Variations and corresponding WT sequences are shown. The patient carries two missense mutations of GMPPB gene (arrows), inherited from his mother (c.332 T > G) and father (c.640G > A), respectively. e Pedigrees

Fig. 2

GMPPB mutants exhibit significantly decreased enzymatic activity. a-b Validation of the designed system for evaluating GMPPB’s enzymatic activity. Absence of Mg²⁺ or substrate mannose-1-p leads to no detectable formation of GDP-mannose (a). The time course of GDP-man production catalyzed by variant doses of GMPPB (b). 0 μg, 5 μg, 10 μg or 20 μg purified GMPPB was used to catalyze the reaction, which was terminated at the indicated time. The reaction mixtures were subjected to Q-ion exchange chromatography, and the amount of GDP-mannose was determined. c V111G mutation causes significantly decreased activity of GMPPB. Purified GMPPB WT, GMPPB V111G, or GMPPB G214S were subjected to activity detection. Compared to the WT, V111G mutation caused significant reduction of GMPPB activity, whereas GMPPB G214 variant retained activity comparable to WT. d Disease-associated GMPPB mutation results in significantly decreased enzymatic activity. 17 reported GMPPB mutants identified in patients were expressed in E.coli and subjected to activity detection. All the tested mutants exhibited significantly compromised activity relative to GMPPB WT. Mean ± SD, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. p values were calculated using one-way ANOVA, Tukey’s multiple comparisons test

Fig. 3

Gmppb knockdown(KD) in Zebrafish causes abnormal development of muscle and motor neurons, movement disability. a Temporal expression of zebrafish gmppb from fertilization to day 3, and gapdh was used as control. b WISH shows the spatial expression pattern of gmppb using a dig-labeled antisense probe. gmppb was ubiquitously expressed in zebrafish embryos from 50%-epi to 24hpf. Scale bar: 250 μm. c Immunoblot of entire zebrafish tissue extracts indicates that gmppb MO injection effectively decreased Gmppb protein level. MO injection was performed at the one cell stage. Actin was used as a loading control. d Expression of myod in zebra fish, with or without gmppb MO injection, determined by WISH. The black rectangles label the position of enlarged views (bottom). Scale bar: 250 μm. e Gmppb KD results in decreased HuC (green) expression. Tg [HuC: GFP] transgenic zebrafish was injected with control MO (NC), or gmppb MO alone or together with mRNA encoding GMPPB. HuC expression was observed at 48 hpf. Scale bar: 100 μmfgmppb mutant larvae display decreased motor ability. Representative images and summarized movement path of zebrafish injected with control MO (NC) or gmppb MO. (Upper). Bar graph summarizes the moving distance and moving speed of zebrafish injected with control MO (NC) or gmppb MO (Lower). Motor ability was monitored in 2 dpf larvae. g. GMPPB is required for motor neuronal development in zebra fish. Tg [hb9: GFP]^ml2 transgenic zebrafish was injected with control MO (NC), or gmppb MO alone or together with mRNA encoding GMPPB at one-cell stage. Top: morphology of CaP axons was observed at 24 and 48hpf; bottom: statistical results of the length of CaP axons. Scale bar: 100 μm. Mean ± SD, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. p values were calculated using one-way ANOVA, Tukey’s multiple comparisons test

Fig. 4

GMPPB activity is required for muscle and neuronal development (a-b) GMPPB V111G mutant with decreased activity fails to rescue axonal phenotype in gmppb MO-injected zebrafish. Morphology of CaP axons from embryos at 24 and 48 hpf that were injected with control MO (NC), or gmppb MO alone or together with mRNA encoding GMPPB WT or its mutants at one-cell stage of the Tg [hb9: GFP]^ml2 transgenic zebrafish embryos. Scale bar: 100 μm (a). Statistical results of the embryos with abnormally branched axons (b). c-d GMPPB V111G mutant fails to rescue muscle defects caused by Gmppb KD. Expression of myod in zebra fish injected with control MO (NC), or gmppb MO alone or together with mRNA encoding GMPPB WT or its mutants. Scale bar: 100 μm (c). Phalloidin staining of filamentous actin (green) in zebra fish injected with control MO (NC), or gmppb MO alone or together with mRNA encoding GMPPB WT or its mutants at one-cell stage. Scale bar: 100 μm (d). e The enzymatic activity of GMPPB mutant positively correlates its ability in rescuing axonal phenotype. Morphology of CaP axons from embryos at 24 and 48 hpf that were injected with control MO (NC), or gmppb MO alone or together with different mRNA at one-cell stage of the Tg [hb9: GFP]^ml2 transgenic zebrafish embryos. Scale bar: 80 μm. Mean ± SD, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. p values were calculated using one-way ANOVA, Tukey’s multiple comparisons test