Impact of PYROXD1 deficiency on cellular respiration and correlations with genetic analyses of limb-girdle muscular dystrophy in Saudi Arabia and Sudan

Abstract

Next-generation sequencing is commonly used to screen for pathogenic mutations in families with Mendelian disorders, but due to the pace of discoveries, gaps have widened for some diseases between genetic and pathophysiological knowledge. We recruited and analyzed 16 families with limb-girdle muscular dystrophy (LGMD) of Arab descent from Saudi Arabia and Sudan who did not have confirmed genetic diagnoses. The analysis included both traditional and next-generation sequencing approaches. Cellular and metabolic studies were performed on Pyroxd1 siRNA C2C12 myoblasts and controls. Pathogenic mutations were identified in eight of the 16 families. One Sudanese family of Arab descent residing in Saudi Arabia harbored a homozygous c.464A>G, p.Asn155Ser mutation in PYROXD1, a gene recently reported in association with myofibrillar myopathy and whose protein product reduces thiol residues. Pyroxd1 deficiency in murine C2C12 myoblasts yielded evidence for impairments of cellular proliferation, migration, and differentiation, while CG10721 (Pyroxd1 fly homolog) knockdown in Drosophila yielded a lethal phenotype. Further investigations indicated that Pyroxd1 does not localize to mitochondria, yet Pyroxd1 deficiency is associated with decreased cellular respiration. This study identified pathogenic mutations in half of the LGMD families from the cohort, including one in PYROXD1. Developmental impairments were demonstrated in vitro for Pyroxd1 deficiency and in vivo for CG10721 deficiency, with reduced metabolic activity in vitro for Pyroxd1 deficiency.

Keywords: CG10721; PYROXD1; exome sequencing; limb-girdle muscular dystrophy.

Figures

Fig. 1.

Genetic analysis of family 1288. A: pedigree showing the affected female (1288-1) and her consanguineous proband, as well as the genotype at the mutated locus (PYROXD1 c.464A>G, p.Asn155Ser) for all family members whose DNA samples were sequenced. B: Sanger sequencing results for PYROXD1 c.464A>G of all family members depicted in the pedigree are shown.

Fig. 2.

Pyroxd1 siRNA knockdown leads to cellular defects in C2C12 myoblasts compared with controls. A: Western blot shows expression of Pyroxd1 protein after transfection with Pyroxd1 siRNA and scrambled siRNA in C2C12 myoblasts. B: these results were quantified via densitometric analysis, n = 3. C: RT-PCR expression analysis was performed on mRNA extracted from Pyroxd1 and scrambled siRNA in C2C12 myoblasts. Data represent the means ± SE from at least three independent experiments, each done in triplicate. Expression levels (2-∆∆CT) are shown relative to an 18S endogenous control. D: proliferation patterns were compared with a CyQUANT DNA quantification kit, with scatter plots representing the mean absorbance ± SE from 24 wells in a 96-well plate. E: a migration assay was performed to create a cell-free zone and migration pattern was observed at 24–72 h. Irregular white lines indicate leading edges of cell migration. F: 10 days after switching to myogenic differentiation medium, a typical microscope field of scrambled siRNA and Pyroxd1 siRNA treated C2C12 cells shows the presence of similar multinucleated myotubes for each group, defined by the presence of at least three nuclei within a cell, with positive desmin staining. G: the scatter plots summarize myoblast fusion index calculations from three independent experiments, each of which included the assessment of five distinct microscope fields. H: Western blot shows the presence of Pyroxd1 in cytoplasmic and nuclear subcellular fractions but not in mitochondrial fractions. C, cytoplasm; M, mitochondria; N, nucleus. I: immunofluorescence of untreated and treated C2C12 cells with antibodies to Pyroxd1 (green) and TOMM 20 (red) shows that Pyroxd1 localizes at the cytoplasm and the nucleus. Bar, 20 µm. ***P < 0.001; ****P < 0.0001; RFU, relative fluorescence units; ns, not significant; scale bar, 20 µm.

Fig. 3.

Functional analyses of C2C12 myoblasts that overexpress human N155S and control PYROXD1. A: confirmation of GFP expression in C2C12 myoblasts that were transfected with human N155S and control PYROXD1 tagged with GFP. DNA quantification was performed using a CyQUANT kit on myoblasts overexpressing N155S vs. control human PYROXD1 (B) and Pyroxd1 siRNA knockdown myoblasts with and without rescue via control human PYROXD1 (C); scatter plots represent the mean absorbance ± SE from 24 wells (B) or 20 wells (C) in a 96-well plate. D: culture dishes were scratched to create cell-free zones, followed by observations of migration patterns at 24–72 h. E: representative photographs of microscope fields obtained 10 days after switching to myogenic differentiation medium shows patterns of multinucleated myotubes that were previously transfected with N155S vs. control PYROXD1, defined by the presence of at least three nuclei within a cell, along with desmin staining patterns. F: the scatter plots summarize myoblast fusion index calculations from three independent experiments, each of which included the assessment of five distinct microscope fields.

Fig. 4.

Metabolic studies comparing Pyroxd1 siRNA knockdown C2C12 myoblasts vs. scrambled siRNA controls indicate a potential impact on mitochondrial function. MTT-based measurement of succinate dehydrogenase activity showed significant differences between Pyroxd1 siRNA knockdown cells vs. controls (A) and between myoblasts overexpressing N155S vs. control human PYROXD1 (B), with scatter plots representing the mean absorbance ± SE (***P < 0.001; ****P < 0.0001). C: intracellular ATP measurements also showed significant differences between Pyroxd1 siRNA knockdown myoblasts vs. controls (****P < 0.0001). Seahorse mitochondrial stress tests were performed to determine the oxygen consumption rate (OCR) (D), the statistical analysis oxygen consumption rate (E), and the extracellular acidification rate (ECAR) (F). The OCR and ECAR data points shown represent values averaged from three independent experiments. G: graphical representation of global OCR vs. ECR activity in Pyroxd1 siRNA-treated C2C12 myoblasts vs. scrambled siRNA-treated myoblasts.

Fig. 5.

Knockdown of the fly homolog of Pyroxd1 is deleterious to the developing organism. A: Homo sapiens pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1 isoform 1: NP_079130.2 (PYROXD1) and its Drosophila melanogaster homolog CG10721-PA: NP_610023 show a high degree of similarity. Arrowhead indicates the p.Asn155 residue that is preserved across these species and is the site of the deleterious mutation. B: ubiquitous downregulation of CG10721 in Drosophila leads to lethality. In the experimental double transgenic progeny (Act5CG4>CG10721 RNAi), the Gal4 transcription factor, expressed under the control of the cytosolic actin5C promoter, in turn drives expression of the dsCG10721 interference construct (in the entire fly). Control siblings, which do not carry the Gal4 transgene and thus do not express the dsCG10721 transgene, develop normally. This was demonstrated in four biological replicates, i.e., progeny from parents isolated at different generation times: n1, 22 control progeny and 0 experimental; n2, 12 control progeny and 0 experimental; n3, 8 control progeny and 0 experimental; and n4, 12 control progeny and 0 experimental. C: two CG10721 RNAi lines were used for the knockdown experiments. The blast result for each interference sequence (orange line at bottom) is shown on the CG10721 gene sequence [schematic obtained from Flybase (16)]. Gray box, noncoding exon; beige box, coding exon. The lethality phenotype was observed using either RNAi line (data not shown for the GD490 fly line).