Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Abstract

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.

Figure 1

Mutations in the MPV17 gene. Schematic representation of the MPV17 gene, comprising eight exons of which exons 2–8 are coding (coding region coloured in orange); the positions of the 4 α-helical transmembrane spanning domains are indicated by red bars; missense/inframe deletion mutations are shown above the schematic and truncating/splicing mutations are shown below the schematic; novel mutations are indicated in blue, previously reported mutations also identified in this study in green and previously reported mutations not identified in this study in black.

Figure 2

Location of missense mutations in the MPV17 protein. Schematic representation of the MPV17 protein, which is localized to the inner mitochondrial membrane; the 4 α-helical transmembrane spanning domains are indicated by red rectangles, and the number of the first and last amino acids of each of these domains is annotated in white; novel missense mutations are indicated in blue, previously reported missense mutations also identified in this study in green and previously reported missense/inframe deletion mutations not identified in this study in grey.

Figure 3

PicoGreen staining of fibroblast cultures. Healthy control fibroblasts exhibit typical bright punctate nucleoid staining when stained with the fluorescent DNA dye PicoGreen (a). Co-staining these cells with the potential sensitive mitochondrial stain, TMRM demonstrated a normal polarized mitochondrial network in all cells (b). Fibroblasts from patient 3 mostly exhibit typical bright nucleoids staining, but some cells (arrow) lack puncta (green in on-line version) because they are depleted of mtDNA, termed mosaic mtDNA depletion (c). Co-staining of these cells with TMRM demonstrates that some of the mtDNA-depleted cells have a depolarized mitochondrial network (d). Fibroblasts grown from patient 17 umbilical cord also show mosaic mtDNA depletion (e), most cells having normal TMRM signal (f) but one depolarized cell is shown (arrow) consistent with mosaic mtDNA depletion. Scale bars=50 μm.