PMC

Photographs of P3. a Low-set ears, b scoliosis, c hand size asymmetry, d arched feet

Identification of PYROXD1 mutations. a Pedigrees of three novel PYROXD1 families and chromatopherograms showing the mutations. b Schematic representation of PYROXD1 and position of known mutations (black) and the novel mutation (red)

Skeletal muscle histopathology. H&E, NADH-TR, and Gomori trichrome staining of transverse muscle section from P1, P2, and P3 revealed similar histological features as fiber-size heterogeneity, fibrosis, rods, and fibers with multiple internalized nuclei (black arrows) and cores (white arrows)

Increased oxidative stress markers. a Western blot and b quantification on muscle extracts from two PYROXD1 patients revealed increased protein levels of HSP70 monomers (70 kDa) and dimers (140 kDa) and glutathione reductase compared with age-matched controls

Skeletal muscle ultrastructure. Electron microscopy on muscle section from P2 and P3 confirmed the presence of cores and rods (white arrows), and revealed glycogen accumulations (black arrow), abnormal mitochondria, and dense osmiophilic bodies (yellow arrow) of unknown origin outside the sarcolemma and within fibres

Protein accumulations in patient muscles. Immuno- and chemical staining of muscle biopsies from P2 and P3 revealed accumulations of the myofibrillar proteins desmin, myotilin, and alpha B crystallin, and of the mitochondrial marker COX, and detected a few fibers expressing foetal myosin, or with positive labelling for the p62 autophagy marker

Characterization of the deep intronic mutation. a The c.1116G > C mutation appears heterozygous on the P3 DNA and homozygous on the RNA. b The PYROXD1 mRNA was strongly reduced in the muscle from P3. c Discriminative PCR on skeletal muscle cDNA revealed the presence of an aberrant amplicon with increased size (transcript 2). d Sequencing of the aberrant transcript 2 showed the inclusion of an additional 110 nt exon with in-frame stop codon (highlighted in black). The intronic mutation (red) reinforces a cryptic donor site (green)