Fig 1: Expression of wild-type MSTO1 rescues cellular phenotypes in MSTO1 patient fibroblasts. Control, P4 and P7 fibroblast cells were transfected with MSTO1-P2A-mCherry or the mCherry empty vector control. Representative images of fibroblasts transfected with MSTO1-P2A-mCherry, for a live cells stained with picogreen and MitoTracker Deep Red, or b fixed cells stained with antibodies against TOMM20 (green, mitochondria) and LAMP1 (blue, lysosomes). Scalebars: 10 µm. Transfected fibroblasts, as identified by cytosolic mCherry signal, were characterized as described above for the following cellular phenotypes: c mitochondrial morphology, d lysosome morphology, e average mtDNA nucleoid size, f mtDNA nucleoid counts, and g relative mtDNA copy number. Student T test was performed as indicated for c, d, f, and g. K–S test was performed to determine statistical significance for e. *p < 0.05, **p < 0.01, ***p < 0.0001
Fig 2: Pathogenic variants in MSTO1 are linked to mtDNA depletion. a Relative mtDNA copy number normalized to the nuclear-encoded 18S gene. Data represent at least three independent biological replicates. b Analysis of mtDNA nucleoid counts per cell from 35 cells for each group. c Quantification of nucleoid sizes in control and patient cells. Data represent average nucleoid sizes from the same cells as in b. Average mtDNA nucleoid size is presented in a violin plot. K–S test was performed to determine statistical significance. d Frequency of nucleoids larger than 0.2 µm2 in all 35 cells quantified per fibroblast line. Student T test was performed as indicated for a, c and d. *p <0.05, **p <0.01, ***p <0.0001
Fig 3: Muscle biopsy, MSTO1 pathogenic variants and pedigrees. a Histology findings from the vastus lateralis muscle biopsy of P7 [p.(Leu450Phe); deletion] at age 20 months include internalized nuclei on hematoxylin and eosin (H&E) staining (white arrow) (i) and variation in fiber size on nicotinamide dinucleotide (NADH) staining (ii) and whorled fibres evident on Gömöri trichrome (inset) (iii) and COX staining (white arrow) (iv). b Muscle biopsy electron microscopy (EM) findings are notable for aggregates of subsarcolemmal mitochondria in both P9 [p.(Tyr478Cys); missing)] (i and ii) and P10 [p.(Asp236Gly); p.(Arg279His)] (iii and iv) and non-specific mitochondrial morphologic abnormalities (variations in mitochondrial shape and size) in P10. c Schematic of new and reported human MSTO1 pathogenic variants. Shown in numbered light blue squares are cDNA exons (RefSeq isoform NM_018116.3 of MSTO1). Corresponding known protein domains are shown in orange (tubulin 3 domain) and beige (Misato segment II tubulin-like domain). Variants written in black text are recessive; the single mutation in red has been previously reported to cause dominantly inherited MSTO1-related disease. The top half of the figure depicts novel variants reported in this publication; the bottom half of the figure depicts variants which have been previously reported. Bolded variants depict previously reported mutations that were also present in our cohort. The dotted line depicts a large deletion (exons 9-14). d Pedigree of two families consistent with recessive inheritance of MSTO1 pathogenic variants
Fig 4: Enlarged lysosomal vacuoles in MSTO1 patient fibroblasts. a Representative confocal images of control and patient cells fixed and stained with antibodies against TOMM20 (red, mitochondria) and LAMP1 (green, lysosomes). Compared to an unaffected control, patient cells contain distinct lysosomal clusters. b Quantification of cells containing enlarged lysosomes in control and patient fibroblasts performed from two independent replicates. Statistical analysis was performed; Student T test, *p <0.05
Fig 5: Characteristics of MSTO1 patient fibroblasts. a Representative confocal microscopy images of control and patient cells. Mitochondrial networks in MSTO1 patient cells are more fragmented and contain fewer but larger mtDNA nucleoids compared to the control cells. Live cells were stained with MitoTracker Red (red, mitochondria) and PicoGreen (green, nuclear and mitochondrial DNA). b Quantification of mitochondrial morphology from control and patient cells performed from three independent replicates. Statistical analysis was performed on the number of cells with partly fragmented mitochondrial morphology in control versus patient cells; Student T test, *p < 0.05, **p < 0.001
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