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.

INTRODUCTION

Limb-girdle muscular dystrophy (LGMD) is a neuromuscular disorder characterized by progressive proximal muscle weakness, accompanied by classic histological findings on muscle biopsy, including fiber size variability, necrosis, regenerating fibers, and inflammation. A large and ever-increasing number of genes have been associated with different types of LGMD, and genotype-phenotype correlations for inherited muscle diseases have become more complex due to the broad sweep of next-generation sequencing. We previously reported a series of consanguineous families affected by LGMD from Saudi Arabia and demonstrated the utility of homozygosity mapping in searching for pathogenic mutations in known associated genes (5).

PYROXD1 is among the newer genes associated with inherited muscle disease, having recently been reported to be associated with early-onset myofibrillar myopathy in families of Turkish and Persian Jewish ancestry (30). PYROXD1 is located at chromosome 12p12.1 and encodes pyridine nucleotide-disulfide oxidoreductase domain-containing protein 1, a protein with a predicted FAD binding site (30), suggesting a possible role in energy metabolism.

As a follow-up to our previously published study of LGMD from Saudi Arabia (5), we recruited an additional 16 families of Arab descent from that country and from Sudan. These families were analyzed via traditional approaches such as immunohistochemistry, Western blotting, and/or Sanger sequencing, followed by exome sequencing for those who remained undiagnosed. This combined approach yielded pathogenic mutations in eight of the 16 families, including one Sudanese family of Arab descent residing in Saudi Arabia who harbored a homozygous missense mutation in PYROXD1. Further analysis of Pyroxd1 in C2C12 myoblasts yielded cellular defects and potentially reduced mitochondrial respiration. Complementary analyses in Drosophila showed that RNA interference (RNAi)-mediated knockdown of CG10721, the fly homolog of Pyroxd1, results in lethality during development.

MATERIALS AND METHODS

Subject recruitment, Sanger sequencing, and whole exome sequencing.

Sixteen families from Saudi Arabia and Sudan with the clinical diagnosis of LGMD were enrolled and analyzed via institutionally approved research protocols at King Saud University College of Medicine, Riyadh, Saudi Arabia; Faculty of Medicine, University of Khartoum, Sudan; Boston Children’s Hospital, Boston, MA; and the University of Florida, Gainesville, FL. Clinical phenotype data and DNA samples were collected. Fifteen of the families have not previously had phenotype or genotype data published. One family (1396) represented a branch of a larger family that was previously found to have a mutation in SGCB, but the individuals from 1396 in the current study had not previously been sequenced (39, 43). Sanger sequencing and whole exome sequencing were performed and analyzed as previously described (34, 35). Allele frequencies were calculated with the Exome Aggregation Consortium (ExAC) database (exac.broadinstitute.org) and the Genome Aggregation Database (gnomAD) (gnomad.broadinstitute.org) (22). Potential pathogenicity was predicted via PolyPhen-2 (genetics.bwh.harvard.edu/pph2) (1). The University of California Santa Cruz Genome Browser (genome.ucsc.edu) was used to determine amino acid conservation across species (19).

Cell culture.

C2C12 myoblasts (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) with 20% fetal bovine serum (Sigma), penicillin (50 units/ml), and streptomycin (50 μg/ml) (Invitrogen). At 90% confluence, growth medium was replaced with low-serum differentiation medium (2% equine serum in DMEM) containing antibiotics. Differentiation medium was replenished daily for 4 days (21, 38).

Pyroxd1 siRNA knockdown and PYROXD1 overexpression.

A cocktail of four Pyroxd1 small interfering RNAs (siRNAs) was transfected into C2C12 cells with DharmaFECT transfection reagent (Dharmacon). The Pyroxd1-specific siRNA sequences were: GGAUAAUGAUUGUCGGGAA, CGAGGGAAAUCCACGUGUA, ACAUUAAGGUCAUCGAAUC, and CCAUAAAGGAUAACGCCAU. Scrambled siRNAs were similarly transfected into C2C12 cells to generate negative controls. Plasmids containing p.Asn155Ser and control PYROXD1 human sequences were a kind gift from Frances Evesson and Sandra Cooper (30). These plasmids were transfected into C2C12 myoblasts via Lipofectamine 3000 (Thermofisher Scientific) and then selected via the administration of gentamicin at 500 μg/ml.

RNA isolation and real-time PCR.

Total RNA was extracted from C2C12 cells with the RNAqueous-4PCR kit (Ambion, Thermo Fisher Scientific) and reverse-transcribed to cDNA with the high-capacity RNA to cDNA kit (Applied Biosystems). RT-PCR of cDNA was performed with the TaqMan Gene Expression Master Mix and TaqMan primer probe set designed for Pyroxd1, with 18S probe sets serving as controls (Thermo Fisher Scientific). Transcript levels were normalized to 18S transcript levels by the ΔΔCT method (38).

Immunoblot studies.

Protein was extracted from C2C12 cells by lysing the myoblasts in RIPA buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 1 mM Na₃VO₄). Cell lysate was spun at 14,000 g at 4°C for 20 min. The total protein content of the supernatant was measured (BCA, Sigma), and 50 μg of the extract was resolved on a 4–12% SDS-polyacrylamide gel (Life Technologies) and then transferred onto a nitrocellulose membrane (20 µm). The membrane was blocked in 5% milk/TBST (0.5% Tween-20, 8 mM Tris-Base, 25 mM Tris·HCl, 154 mM NaCl) and then probed with antibodies to Pyroxd1 at a 1:250 dilution (Abcam) and Gapdh at a 1:1,000 dilution (Cell Signaling Technologies). The membrane was incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by chemiluminescence (Thermo Scientific).

Subcellular fractionation assay.

C2C12 myoblasts were grown to 98% confluence in 10 cm dishes (~1 × 10⁷ cells/dish). Cytoplasmic and nuclear fractions were isolated from these cells as previously described (28). Mitochondria were isolated as previously described (33). Immunoblot studies were performed on the subcellular protein fractions by the above protocol and the following antibodies for the loading controls: anti-Gapdh antibodies (Cell Signaling) for the cytoplasm, anti-Histone 3 antibodies (Cell Signaling) for the nucleus, and anti-VDAC antibodies (Sigma) for the mitochondria.

Immunofluorescence and myoblast fusion.

C2C12 cells were cultured sparsely on coverslips, washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde, and then permeabilized with 0.1% Triton X-100 in PBS. After being blocked with 10% fetal bovine serum in PBS, the cells were incubated with primary anti-Pyroxd1 (Abcam), Tomm 20 (Sigma), and anti-Desmin antibodies (Abcam) and then with secondary Alexa-488 conjugated goat anti-rabbit antibody or Alexa-568 conjugated goat anti-mouse antibody (Life Technologies). Coverslips were mounted with DAPI. Confocal images were visualized on an Olympus BX43 upright microscope. The myoblast fusion index was calculated as the ratio of the number of nuclei inside myotubes compared with the total number of nuclei × 100 at day 10 of myogenic differentiation via ImageJ software as previously described (38).

Cell functional assays.

Cell proliferation and migration assays were performed and analyzed as previously described (38) on C2C12 myoblasts transfected with Pyroxd1 and scrambled siRNA, as well those transfected with p.Asn155Ser and control PYROXD1 human sequences. The CyQUANT DNA quantification assay was used to assess cell proliferation, as this assay correlates well with cell counts (38).

General metabolic assay.

Measurements of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction were used to assess cellular succinate dehydrogenase activity (23, 27). For one experiment, C2C12 myoblasts transfected with Pyroxd1 and scrambled siRNA and untransfected cells were cultured in 96-well plates for 24, 48, and 72 h. A complementary experiment was performed via transfection of p.Asn155Ser and control PYROXD1 human sequences. MTT (Sigma) was dissolved in PBS at 5 mg/ml and added to each well and incubated at 37°C for 4 h. After 4 h, dimethyl sulfoxide was added to the wells, followed by incubation for 30 min and measurement of absorbance at 570 nm. This experiment was performed three times.

Measurement of intracellular ATP.

Intracellular ATP level was measured with the luminescence ATP detection assay (ATPlite; PerkinElmer, Waltham, MA), which is based on the production of light caused by the reaction of ATP with luciferase and D-luciferin. The emitted light is proportional to the ATP concentration. Cells were seeded in 96-well plates, and then 50 µl of cell lysis solution was added to lyse the cells and release ATP. Luciferase was added for 5 min, followed by the addition of D-luciferin for another 5 min, then 10 min of dark adaptation. Luminescence was measured with a microplate reader (Molecular Devices). The ATP standard curve was generated by plotting signal vs. ATP concentrations. The signal for the unknown sample was obtained via linear regression analysis. The ATP measurement was then normalized to protein concentration (24). This experiment was performed twice.

Cellular respiration assay.

Cellular respiration, represented by the oxygen consumption rate (OCR), and glycolysis, represented by the extracellular acidification rate (ECAR), were measured 48 h post-siRNA transfection with the XF-96 Analyzer (Seahorse Bioscience) (29). The XF assay medium (${\text{HCO}}_3^{-}$-free modified DMEM, Seahorse Bioscience) was supplemented with 25 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate and with further additions relevant to the experiment. The pH was adjusted to 7.4 at 37°C. For C2C12 myoblast experiments, we seeded 10,000 cells per well of siRNA transfected cells after 48 h of transfection. The plate incubated overnight, allowing the cells to attach in the respective culture medium as a monolayer. Mitochondrial respiration testing was then performed by sequential additions of 1 µM oligomycin, 2.0 µM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), and 0.5 µM rotenone/antimycin A. The following mitochondrial parameters were measured: basal respiration, basal mitochondrial respiration (basal cellular respiration minus nonmitochondrial respiration), ATP turnover-driven respiration (basal respiration minus oligomycin-inhibited respiration), maximal respiratory capacity (maximal uncoupled respiration minus nonmitochondrial respiration), spare respiratory capacity (maximal uncoupled respiration minus basal respiration), proton leak (oligomycin-inhibited respiration minus nonmitochondrial respiration), and nonmitochondrial respiration (rotenone/antimycin A-inhibited respiration). The OCR results were expressed in pmol (O₂)/minute (time) × µg (protein). ECAR results were expressed in mpH·min⁻¹·µg⁻¹. This experiment was performed three times.

Drosophila studies.

Two UAS-dsCG10721 RNAi stocks (FBst0482296: P{KK102834}VIE-260B and FBst04599301: w1118; P{GD490}v33098) were obtained from the Vienna Drosophila RNAi Center (VDRC, Vienna, Austria). The interference construct is expressed under the control of the well-characterized Gal4/UAS binary system (6). The Gal4 driver fly line FBst0004414: y1w*;P{Act5C-GAL4}25FO1/CyO was obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN). All stocks were maintained at 25°C in a 12 h light-12 h dark cycle on standard Drosophila medium. To generate Drosophila that downregulate CG10721/Pyroxd1, the VDRC UAS-dsCG10721 homozygous flies were crossed with the Gal4 driver flies at 29°C, the optimal temperature for the Gal4/UAS system. The experimental progeny carried both Gal4 and UAS transgenes, while isogenic siblings (that share the same set of parents and develop in the same vial) bore only the UAS transgene and were thus used as control flies for phenotypic analyses.

Statistics.

All data were expressed as means ± SE. Statistical analyses were performed with GraphPad Prism 5 (GraphPad Software). Unpaired t-tests were performed between groups. A P value <0.05 was considered statistically significant.

RESULTS

Clinical phenotypes.

Highlights of the clinical presentations of the families found to have pathogenic mutations are noted in Supplemental Table S1. (The online version of this article has supplemental material.) The family found to have a PYROXD1 mutation is of particular interest, and the proband’s clinical presentation is as follows. A 37 yr old Sudanese female of Arab ancestry who lives in Saudi Arabia presented with progressive muscle weakness that began at the age of 9 yr. Initial symptoms included excessive falling while running, with slowly progressive weakness. She had difficulty navigating stairs by 18 yr of age but was able to complete her university education. After marrying at age 30 yr, she gave birth to an apparently unaffected girl 1 yr later and then a boy after another 2 yr, both via Cesarean section due to failure to progress. At the age of 33½ yr, she tripped and fractured her right femur; this fracture was managed by casting. She was noted to be significantly weaker following removal of the cast but continued to ambulate with the assistance of a walker. She lost ambulation at 37 yr of age. She was the first child of distantly related parents (Fig. 1A) and was born at term with no neonatal complications. Early developmental milestones were normal, and she began to walk by the age of 15 mo. General examination was unremarkable at 37 yr. On neurological examination, she had significant proximal symmetrical muscle weakness and wasting, along with calf muscle pseudohypertrophy. By that time she was nonambulatory, but she was able to sit erect in a chair, push her manual wheelchair, and eat and drink independently. Serum creatine kinase was 74 U/l (reference range 21–232). The serum alamine aminotransferase level was slightly elevated at 75 U/l (reference range 30–65). Other laboratory investigations were unremarkable, including electrolytes, blood urea nitrogen, uric acid, triglycerides, and cholesterol. Electromyography revealed myopathic features. Muscle biopsy was not performed. At her most recent follow-up evaluation, she was 46 yr old, and her motor function remained generally stable.

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.

Genetic analysis.

The findings from mutation analysis are summarized in Table 1. Sanger sequencing of common LGMD genes identified pathogenic mutations in FKRP (family 1304), SGCA (families 1307, 1308, and 1310), and SGCB (family 1396). The SGCA mutation in family 1307 has not previously been published. Whole exome sequencing on seven of the remaining families revealed pathogenic mutations in three additional families. One was a previously unpublished mutation in SGCG (family 1398), and the other was a previously unpublished mutation in COL6A1, which is typically associated with Bethlem myopathy and Ullrich congenital muscular dystrophy (family 1306). Most notably, a recently reported pathogenic PYROXD1 mutation, c.464A>G, p.Asn155Ser (30), was identified on whole exome sequencing of family 1288. Sanger sequencing of this region on DNA samples from seven family members across three generations, including the proband, unaffected parents, siblings, and children confirmed the presence of this homozygous mutation in the proband and cosegregation with the phenotype (Fig. 1B and Table 1). Sanger sequencing of all coding exons of PYROXD1 on DNA samples obtained from the probands of the eight families in the cohort without genetic diagnoses did not yield additional mutations (Supplemental Table S2). The allele frequency of the PYROXD1 c.464A>G, p.Asn155Ser mutation was found to be 0.00007157 in ExAC and 4.624e-5 in gnomAD, while PolyPhen-2 predicted that the mutation would be damaging, with a specificity score of 1.0. The Asn155 residue is conserved across numerous species from zebrafish to humans, according to the UCSC Genome Browser.

Table 1.

Genetic analysis for the 10 Saudi Arabian and Sudanese families with genetic diagnoses

Family	Gene/Locus	Mutation	Inheritance	(SNP) (References)	Exome Sequencing	Sanger Sequencing	Segregation of Mutation
1304	FKRP	NM_024301.4 c.941C>T; NP_077277.1 p.Thr314Met; hg19 chr19:47259648	homozygous recessive (consanguineous)	(rs398124395) (5, 26)	not done	proband (♀) and parents	homozygous in proband, heterozygous in both parents
1306	COL6A1	NM_001848.2 c.128A>C; NP_001839.2 p.Asp43Ala; hg19 chr21:47402328	homozygous recessive (consanguineous)	(rs786205555) (Developmental Genetics Unit, King Faisal Specialist Hospital & Research Centre)	proband (♀) and parents	proband, parents, affected brother and unaffected sister	homozygous in proband and affected brother; heterozygous in both parents and unaffected sister
1307	SGCA	NM_000023.3 c.241C>T; NP_000014.1 p.Arg81Cys; hg19 chr17:48244776	homozygous recessive (consanguineous)	novel (rs398123098) (Emory Genetics laboratory)	not done	proband (♂), parents and 3 unaffected siblings	homozygous in proband, heterozygous in both parents and unaffected brother; absent in unaffected brother and unaffected sister
1308	SGCA	NM_000023.3 c.220C>T; NP_000014.1 p.Arg74Trp; hg19 chr17:48244755	homozygous recessive (consanguineous)	(rs757888349) (10, 12, 15, 18, 25, 45)	not done	proband (♀), parents and five unaffected siblings	homozygous in proband, heterozygous in both parents, unaffected brother and two unaffected sisters; absent in unaffected brother and unaffected sister
1310	SGCA	NM_000023.3 c.226C>T; NP_000014.1 p.Leu76Phe; hg19 chr17:48244761	homozygous recessive (consanguineous)	(2, 10)	not done	proband (♀), parents and four unaffected siblings	homozygous in proband, heterozygous in both parents and unaffected sister; absent in two unaffected sisters and unaffected brother
1396	SGCB	NM_000232.4 c.112_113del CT; NP_000223.1 p.Ser38X; hg19 chr4:52899400–52899901	homozygous recessive (consanguineous)	(45)	not done	proband (♂), mother and two other affected siblings	homozygous in proband, 1396-2 and 1396-3. heterozygous in mother (1396-4)
1398	SGCG	NM_000231.2 c.212T>C; NP_000222.1 p.Leu71Ser; hg19 chr13:23808516	homozygous recessive (consanguineous)	(rs143009120)	proband only (♀)	proband, parents, two unaffected siblings	homozygous in proband, heterozygous in both parents, unaffected brother and unaffected sister
1288	PYROXD1	NM_024854.3 c.464A>G; NP_079130.2 p.Asn155Ser; hg19 chr12:21605064	homozygous recessive (consanguineous)	(rs781565158) (30)	proband only (♀)	proband, parents, two unaffected siblings and two children	homozygous in proband, heterozygous in both parents, unaffected sister, unaffected son and unaffected daughter; absent in unaffected brother

Pyroxd1 deficiency impairs proliferation, migration, and differentiation in murine myoblasts; Pyroxd1 localizes to both nucleus and cytoplasm but not mitochondria.

Pyroxd1 expression in C2C12 myoblasts was knocked down with siRNA; decreased levels were confirmed via Western blot (Fig. 2, A and B) and RT-PCR (Fig. 2C) analyses. Pyroxd1 deficiency resulted in impaired proliferation (determined with the CyQUANT DNA quantification assay) (Fig. 2D) and migration (Fig. 2E) of C2C12 myoblasts, along with desmin expression (Fig. 2F) and myoblast fusion (Fig. 2G). An adhesion assay did not show a significant difference between the groups (data not shown). On subcellular fractionation assays of protein derived from C2C12 myoblasts (Fig. 2H) and immunofluorescence studies of C2C12 myoblasts (Fig. 2I), Pyroxd1 localized to both the nucleus and cytoplasm, but not mitochondria. Pyroxd1 siRNA-treated myoblasts showed reduced staining of Pyroxd1 compared with scrambled siRNA treated cells (Fig. 2I).

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.

Overexpression of PYROXD1 N155S mutant impairs proliferation, migration, and differentiation of murine myoblasts.

Transfection of C2C12 myoblasts with N155S mutant and control human PYROXD1 was confirmed (Fig. 3A). Expression of mutant N155S in C2C12 myoblasts was associated with reduced proliferation (Fig. 3B) that was rescued by cotransfection of control human PYROXD1 (Fig. 3C). Expression of mutant N155S was also associated with reduced migration (Fig. 3D) and differentiation (Fig. 3, E and F) of those cells compared with controls.

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.

Pyroxd1 deficiency is associated with reduced mitochondrial metabolic activity in C2C12 myoblasts.

Pyroxd1 siRNA knockdown C2C12 myoblasts (Fig. 4A) and myoblasts overexpressing human N155S mutant PYROXD1 (Fig. 4B) demonstrated diminished complex II succinate dehydrogenase activity (MTT assay) compared with controls. The Pyroxd1 siRNA knockdown C2C12 myoblasts were also found to have reduced intracellular ATP content (Fig. 4C) compared with controls. Examination of cellular respiration measured via the OCR demonstrated lower respiration in Pyroxd1 siRNA knockdown myoblasts compared with scrambled siRNA myoblasts across all variables except spare respiratory capacity (Fig. 4, D and E). Measurement of glycolysis via the ECAR (Fig. 4F) demonstrated a trend of lower activity in the Pyroxd1 siRNA knockdown myoblasts compared with scrambled siRNA myoblasts that did not reach statistical significance. An analysis of global OCR and ECAR activity suggests that both are diminished in the Pyroxd1 siRNA knockdown myoblasts compared with scrambled siRNA myoblasts (Fig. 4G).

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.

Expression of dsCG10721 RNAi in the developing fly results in reduced viability.

The Drosophila homolog of PYROXD1 is CG10721, with conservation of the residue affected by the mutation in family 1288 (Fig. 5A). Flies that are reared at 29°C and ubiquitously express a CG10721 RNAi construct arrest during development (i.e., fail to emerge from their pupal cases) (Fig. 5B). The lethality phenotype is recapitulated in a second CG10721 RNAi mutant fly line (Fig. 5C shows the targets of the two RNAi oligonucleotides, viability data not shown).

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).

DISCUSSION

The diagnostic yield of 50% from this Saudi Arabian and Sudanese cohort is in line with previous reports (3, 14, 17, 34). The initial diagnostic screen was based on clinical presentations. Sanger sequencing identified causative mutations in SGCA, SGCB, and FKRP. Exome sequencing yielded additional mutations in SGCG, COL6A1, and PYROXD1. Previous reports suggested that mutations in DYSF, CAPN3, FKRP, and the sarcoglycans are common causes of LGMD in Saudi Arabia (5, 26). One of the Sudanese families (1396) harbors an SGCB mutation and represents one of the earliest reported families with severe childhood autosomal recessive muscular dystrophy, the common phenotype of LGMD in North Africa and the Arabian Peninsula (39–43). Historically, this family was documented to have migrated from central Saudi Arabia to the Sudan, crossing the Red Sea, in the 12th and 13th centuries (44). Similarly, an ancient founder LAMA2 gene mutation (c.3924+2T>C) that causes congenital muscular dystrophy type 1A was found in both Saudi Arabian and Sudanese patients (9).

Similarities between family 1288 and the previously reported ones with PYROXD1 mutations (30) include the slow progression of proximal and symmetrical muscle weakness, accompanied by normal serum creatine kinase levels. However, the proband had a later age of onset compared with most of the previously reported cases. Additionally, the proband in the current study did not develop cardiac or respiratory complications at any point in her course, in contrast to the affected individuals in the previous study. Thus, family 1288 was considered to have an LGMD phenotype, albeit without muscle pathology confirmation.

Deficiency of Pyroxd1 and expression of mutant PYROXD1 cause defects in myoblast proliferation, migration, and differentiation. The differentiation assay revealed that there is a deficiency in myotube fusion as well as desmin expression, which correlates with data from an earlier study (30). The lethality phenotype seen when CG10721 is knocked down in Drosophila confirms the importance of PYROXD1 and its homologs in development across multiple species.

PYROXD1 is expressed across a range of tissue types in humans, including skeletal muscle (11), and is conserved across many species (7, 13). It was only recently identified as a class I pyridine nucleotide-disulfide oxidoreductase (PNDR) that localizes to the nuclei and the sarcomeric/sarcoplasmic compartments of myofibers (30). We have confirmed the previously described subcellular localization. The exact cellular function of this enzyme in muscle remains obscure at this time. PYROXD1 has a putative FAD binding site, and FAD/FADH plays an important role in the mitochondrial electron transport chain (4). The Asn155 residue is predicted to lie in the pyridine nucleotide-disulfide oxidoreductase catalytic site; thus, a mutation at that location could impact FAD cofactor activity (30). Analogously, a single missense mutation has been reported to impair FAD cofactor binding of the thiol oxidase Erv1 (8). A different PNDR, depH, relies on FAD to form disulfide bonds in bacteria (47), thus further studies of PYROXD1 may involve an examination of whether it performs a similar function. Our data suggest that PYROXD1 deficiency is associated with lower rates of metabolic activity in C2C12 myoblasts, consistent with the finding of reduced proliferation in those cells. Future studies could include the characterization of mitochondrial quantity and morphology in either patient muscle biopsy samples or models of PYROXD1 deficiency.

It is unusual for enzyme defects to be associated with LGMD, though a classic example of such an enzyme is CAPN3 (calpain 3), associated with LGMD type 2A (36). Mitochondrial impairments are becoming recognized as important components of the disease mechanisms of certain muscular dystrophies, including LGMDs. Examples include defects in mitochondrial ATP synthesis in a mouse model of Duchenne muscular dystrophy (DMD) (32, 37) and LGMD type 2A (20), electron transport chain deficiencies in LGMD type 2B (dysferlinopathy) (46), and the reduced mitochondrial biogenesis observed in LGMD type 2D (α-sarcoglycanopathy) (31). A link between the structural nature of many of the proteins associated with DMD and the LGMDs, usually localizing at the sarcolemma, and mitochondria may lie in the impact on nitric oxide synthase, a component of the dystrophin-associated protein complex (31).

A limitation of the current study is the lack of conclusive data regarding the mechanism of the metabolic changes that have been identified. Further studies will be needed to determine whether the decreased metabolic activity leads to reduced proliferation or conversely.

In summary, both the overall cohort and family 1288 are notable, and the findings in the latter broaden the range of phenotypes that may be seen with PYROXD1 mutations. The enzymatic nature of PYROXD1 is unusual for a protein associated with LGMD, and further investigations will help explain what connections may exist between PYROXD1 and other muscle disease genes whose protein products play more of a structural role.

GRANTS

This study was funded by National Institutes of Health (NIH) Grant R01 NS-080929 (M. Saha, H. M. Reddy, P. B. Kang) and the Bernard F. and Alva B. Gimbel Foundation (L. M. Kunkel). Exome sequencing and analysis was provided by the Broad Institute of MIT and Harvard that was supported by NIH Grant U54 HG-003067 (Eric Lander). M. A. Salih was supported by the Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia via research group project number RGP-VPP-301.

DISCLOSURES

Dr. MacArthur is a founder with equity holdings in Goldfinch Bio. Dr. Kunkel is on the scientific advisory boards of Sarepta Therapeutics and Summit Therapeutics. Dr. Kang holds a grant from the National Institute for Neurological Disorders and Stroke of the NIH, consults for AveXis, is a co-investigator for Catabasis, receives royalties from Springer for a textbook, serves as an associate editor for Muscle & Nerve and thus receives honoraria from Wiley, and has authored chapters for UpToDate and thus receives honoraria from Wolters Kluwer.

AUTHOR CONTRIBUTIONS

M. Saha, I.D., and P.B.K. conceived and designed research; M. Saha, M.D.J., S.M., K.-A.C., S.S.-H., S.A.R., and I.D. performed experiments; M. Saha, H.M.R., M. Salih, E.E., M.D.J., S.M., K.-A.C., S.S.-H., S.A.R., M.H.H., M.M.M., A.A.H., M.A.E., M.L., E.V., D.G.M., C.A.P., I.D., and P.B.K. analyzed data; M. Saha, H.M.R., M.D.J., S.M., K.-A.C., S.S.-H., S.A.R., M.L., D.G.M., L.M.K., C.A.P., I.D., and P.B.K. interpreted results of experiments; M. Saha, H.M.R., and I.D. prepared figures; M. Saha, H.M.R., I.D., and P.B.K. drafted manuscript; M. Saha, H.M.R., M. Salih, E.E., M.L., C.A.P., I.D., and P.B.K. edited and revised manuscript; M. Saha, H.M.R., M. Salih, E.E., M.D.J., S.M., K.-A.C., S.S.-H., S.A.R., M.H.H., M.M.M., A.A.H., M.A.E., M.L., E.V., D.G.M., L.M.K., C.A.P., I.D., and P.B.K. approved final version of manuscript.