Biallelic Variants in PYROXD2 Cause a Severe Infantile Metabolic Disorder Affecting Mitochondrial Function

Pyridine Nucleotide-Disulfide Oxidoreductase Domain 2 (PYROXD2; previously called YueF) is a mitochondrial inner membrane/matrix-residing protein and is reported to regulate mitochondrial function. The clinical importance of PYROXD2 has been unclear, and little is known of the protein’s precise biological function. In the present paper, we report biallelic variants in PYROXD2 identified by genome sequencing in a patient with suspected mitochondrial disease. The child presented with acute neurological deterioration, unresponsive episodes, and extreme metabolic acidosis, and received rapid genomic testing. He died shortly after. Magnetic resonance imaging (MRI) brain imaging showed changes resembling Leigh syndrome, one of the more common childhood mitochondrial neurological diseases. Functional studies in patient fibroblasts showed a heightened sensitivity to mitochondrial metabolic stress and increased mitochondrial superoxide levels. Quantitative proteomic analysis demonstrated decreased levels of subunits of the mitochondrial respiratory chain complex I, and both the small and large subunits of the mitochondrial ribosome, suggesting a mitoribosomal defect. Our findings support the critical role of PYROXD2 in human cells, and suggest that the biallelic PYROXD2 variants are associated with mitochondrial dysfunction, and can plausibly explain the child’s clinical presentation

Generation of <i>FRG1</i> and <i>FRG1/FHL1</i> mice.

<p>(A) Immunoblot analysis of protein expression in the tibialis anterior, quadriceps, triceps and trapezius muscles from wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice. HA-tagged FHL1 was detected using a HA-specific antibody; β-tubulin immunoblotting and ponceau red staining of membranes were used as loading controls. (B) Relative FRG1 and FHL1 expression levels in the tibialis anterior, quadriceps, triceps and trapezius muscles from wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice. Protein expression was quantified using densitometry. Quantitative RT-PCR analysis of FRG1 (C) and FHL1 (D) mRNA in muscles from wild type, <i>FRG1</i> and <i>FRG1/FHL1</i> mice. Data represent the mean from n≥4 mice/genotype; *p&lt;0.05, **p&lt;0.005, ***p&lt;0.001 determined by two-tailed student’s T-test.</p

C2C12 myoblasts overexpressing FRG1 exhibit a fusion defect.

<p>(A) Immunoblot analysis of FRG1 expression in C2C12 myoblasts expressing HA-vector or HA-FRG1. HA-tagged FRG1 was detected using a HA-specific antibody. Clones HA-FRG1 13 and HA-FRG1 16 were selected for further analysis. Positive control represents HA-FRG1 transfected COS1 cells. (B) qRT-PCR analysis of FRG1 mRNA levels in undifferentiated HA-FRG1 myoblasts relative to HA-vector control myoblasts. Data represent the mean +/- SEM from n = 3 independent experiments; *p&lt;0.05. (C) Representative images of C2C12 myoblasts expressing either HA-vector control or HA-FRG1 as indicated, following 96 hours differentiation and stained with the differentiation marker MHC (red) and ToPro 3-iodide to detect nuclei (blue). (D-F) Several parameters were quantified to assess the efficiency of myoblast differentiation; (D) Frequency of MHC-positive myoblasts and myotubes containing 1, 2, 3, 4 or ≥5 nuclei. Data represent the mean ± SEM from n = 3 independent experiments;***p&lt;0.0005 determined by two-way ANOVA with Tukey’s multiple comparisons test; (E) Average number of nuclei per myotube; (F) Fusion index; the percentage of total nuclei localized within MHC-positive myotubes; (G) Differentiation index; the percentage of total nuclei localized within MHC-positive cells (myocytes and myotubes); (H-I) Relative myogenin and (J-K) MHC expression in HA-vector <i>versus</i> HA-FRG1 expressing myoblasts during 0–96 hours differentiation. Immunoblotting for β-tubulin and staining membranes with ponceau red were used as a loading control. Myogenin and MHC expression were quantified using densitometry. Data for (B), (E-F), (I) and (K) represent the mean ± SEM from n = 3 independent experiments; *p&lt; 0.05 determined by two-tailed Student’s T-test. Scale bars = 50μm.</p

FHL1 reduces muscle wasting in dystrophic <i>FRG1</i> mice.

<p>(A) Representative X-ray images of the spine of mice from the indicated genotypes. (B) Whole body weight in 6-week-old mice; wild-type (n = 10); <i>FRG1</i> (n = 6); <i>FRG1</i>/<i>FHL1</i> (n = 9); and 12-week-old mice; wild-type (n = 11); <i>FRG1</i> (n = 12); <i>FRG1/FHL1</i> (n = 14). (C) Representative image of skinned hind limbs from <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice. Arrows point to the quadriceps muscle to show the difference in muscle mass between the <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice. (D) Representative images of various muscles dissected from wild type, <i>FRG1</i> and FRG1/<i>FHL1</i> mice. (E) Relative muscle weight from 6-week-old mice and 12-week-old mice. Data represents an average of the combined weights from 4 muscle groups (n = 6–10 mice per genotype for the tibialis anterior, quadriceps, triceps and trapezius) and is expressed relative to wild type muscle weight. Data represent the mean ± SEM; *p&lt; 0.05; **p&lt;0.005; ***p&lt;0.0005 determined by two-tailed Student’s T-test.</p

FHL1 reduces fibrosis and fat deposition in dystrophic <i>FRG1</i> mice.

<p>Representative images of transverse sections of (A) quadriceps and (B) trapezius muscle from 12-week-old wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice stained with Masson’s trichrome to detect fibrosis within muscle. The percentage area of fibrosis staining in muscle was quantified from wild type (n = 4–6), <i>FRG1</i> (n = 4–6) and <i>FRG1</i>/<i>FHL1</i> (n = 5–6) mice. Representative images of transverse muscle sections from the (C) quadriceps and (D) trapezius muscle stained with Oil Red O to detect fat deposits within muscle. The percentage area of fat deposition in muscle was quantified in wild type (n = 3–4), <i>FRG1</i> (n = 4) and <i>FRG1</i>/<i>FHL1</i> (n = 4) mice. Data represent the mean ± SEM; *p&lt;0.05; **p&lt;0.005; ***p&lt;0.0005 determined by two-tailed Student’s T-test. Scale bars = 100μm.</p

FHL1 improves muscle pathology in dystrophic <i>FRG1</i> mice.

<p>Representative images of transverse muscle sections from the triceps (A) or quadriceps (D) muscles of 6-week-old wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice stained with H&amp;E. Boxed region indicates area shown in high magnification image inset. Mean myofiber diameter from the triceps (B) and quadriceps (E) was measured for wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice. Histograms showing the frequency of individual muscle fiber diameters from the triceps (C) or quadriceps (F) of wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice. 500–1000 muscle fibers were measured per muscle for each mouse; Wild type (n = 3 mice), <i>FRG1</i> (n = 4 mice) and <i>FRG1</i>/<i>FHL1</i> (n = 4 mice). Data represent mean ± SEM; *p&lt;0.05; **p&lt;0.005 determined by two-tailed Student’s T-test. In (C) and (F), asterisks in <i>FRG1</i> histograms indicate significant differences between <i>FRG1</i> and wild type mice; Asterisks in <i>FRG1</i>/<i>FHL1</i> histogram indicate significant differences between <i>FRG1</i>/<i>FHL1</i> and <i>FRG1</i> mice. Scale bars = 100μm.</p

FHL1 increases the proportion of muscle fibers with centralized nuclei in <i>FRG1</i> mice.

<p>Representative images of transverse sections of triceps (A) or quadriceps (B) muscles from 12-week-old <i>FRG1</i> and <i>FRG1/FHL1</i> mice stained with H&amp;E. Arrows indicate myofibers with centralized nuclei, an indicator of myoblast fusion <i>in vivo</i>. Scale bars = 100μm. For both triceps and quadriceps muscles the percentage of muscle fibers with centralized nuclei was quantified for wild type (n = 3), <i>FRG1</i> (n = 4) and <i>FRG1</i>/<i>FHL1</i> (n = 4) mice. The subset of these fibers containing multiple centralized nuclei was further quantified at 12 weeks of age. Data represent the mean ± SEM; ns not significant, *p&lt;0.05; **p&lt;0.005; ***p&lt;0.0005 determined by two-tailed Student’s T-test.</p

FHL1 does not alter satellite cell number or markers of satellite cell activation (MyoD) or differentiation (myogenin) in the triceps of <i>FRG1</i> mice.

<p>(A) Transverse muscle sections from the triceps of wild type, <i>FRG1</i> and <i>FRG1/FHL1</i> mice (aged 6- and 12-weeks) were co-stained with a satellite cell specific marker (pax7) and DAPI to detect nuclei. Arrows indicate pax7+ satellite cells. Boxed region indicates area shown in high magnification image inset. Scale bars = 100μm. The number of pax7+ satellite cells per 100 myofibers was counted for the triceps in mice aged (B) 6-weeks (<i>FRG1</i> n = 3 and <i>FRG1/FHL1</i> n = 3) and (C) 12-weeks (<i>FRG1</i> n = 4 and <i>FRG1/FHL1</i> n = 4). Quantitative RT-PCR analysis of pax7 (D- 6 weeks, E- 12 weeks) MyoD (F- 6 weeks, G- 12 weeks) and myogenin (H- 6 weeks, I- 12 weeks) mRNA in wild type, <i>FRG1</i> and <i>FRG1/FHL1</i> (n = 7 mice/genotype) triceps muscle. Data represent the mean ± SEM; ns not significant; *p&lt;0.05; **p&lt;0.001 determined by two-tailed Student’s T-test.</p

FHL1 enhances myoblast fusion in <i>FRG1</i> mice.

<p>(A) Representative images of longitudinal sections of triceps muscle from 12-week old wild type, <i>FRG1</i> and <i>FRG1/FHL1</i> mice co-stained for dystrophin to outline the muscle fiber membrane and DAPI to detect nuclei. Boxed region indicates area shown in high magnification image inset. Scale bars = 100μm. (B) The number of nuclei per mm of muscle fiber was counted as a measure of myoblast fusion at 12 weeks (wild type n = 3, <i>FRG1</i> n = 4, <i>FRG1/FHL1</i> n = 4). Data represent the mean ± SEM; ns not significant, *p&lt;0.05determined by two-tailed Student’s T-test.</p

<i>FRG1</i> myoblasts exhibit a fusion defect that is rescued in <i>FRG1</i>/FHL1 myoblasts.

<p>Quantitative RT-PCR analysis of human FRG1 (A) and human FHL1 (B) mRNA in primary mouse myoblasts isolated from wild type mice, and two <i>FRG1</i> (1 and 2) and two <i>FRG1/FHL1</i> (1 and 2) mice. Data represent the mean +/- SEM of n = 3 independent experiments and was standardized to GAPDH and expressed relative to control wild type myoblasts. *p&lt;0.05. Representative images of primary myoblasts from wild type, <i>FRG1</i> and <i>FRG1</i>/<i>FHL1</i> mice following 48 hours (C) or 96 hours (E) differentiation. Cultures were stained with the differentiation marker MHC (green) and DAPI for detection of the fusion index; the percentage of total nuclei localized within MHC-positive myotubes after 48 hours (D) and 96 hours (F) differentiation. Data represent the mean ± SEM from n = 3 independent experiments; wild type (n = 1); <i>FRG1</i> (n = 2); <i>FRG1</i>/<i>FHL1</i> (n = 2); ns not significant, **p&lt;0.005, ***p&lt;0.0001 determined by two-tailed Students T-test. Scale bars = 100μm.</p