Variants in the Oxidoreductase PYROXD1 Cause Early-Onset Myopathy with Internalized Nuclei and Myofibrillar Disorganization

Abstract

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.

Figure 1

Clinical Features of PYROXD1-Related Myopathy (A) Pedigrees of the five affected families. (B) Posterior view of A-II1 (i) demonstrating generalized reduction of muscle bulk and prominent scapulae. Hands of A-II2 (ii) demonstrating marked wasting of the thenar and hypothenar eminence. Foot of A-II1 (iii) showing pes cavus and skin discoloration. C-II1 (iv) demonstrating hyperextension of the elbow and reduced muscle bulk. C-II1 (v) wrist hyperextension. C-II2 (vi) hand and wrist hyperextension. (C) Axial T1 muscle MRI of calf and thigh of individuals A-II2, B-II2, B-II3, and C-II2. In all individuals, the thigh shows generalized reduction in muscle, atrophy, and fatty marbling. There is relative sparing of rectus femoris relative to vastus lateralis. The calf of individuals in family B is more mildly affected. (D) Top: Agarose gel of PCR products of fibroblast cDNA from two affected siblings from family A (left) and family C (right). For both families, sequencing of the lower band established in-frame skipping of exon 3 in family A and exon 4 in family C. Sequencing of the upper band in family A identified only transcripts bearing the maternal c.1116 G>C (p.Gln372His) missense variant. Sequencing of the upper band in family C identified only transcripts bearing the maternal c.464A>G (p.Asn155Ser) variant. Bottom: Schematic of PYROXD1 exon skipping events in family A and family C (not to scale). (E) Sequencing chromatogram of the total PCR mixture using exon 1 forward and exon 12 reverse primers. Family A shows equal peak heights for the paternal wild-type and maternal missense variant c.1116 G>C (p.Gln372His) (red asterisks), suggesting approximately equal abundance of exon 3 skipped transcripts and missense c.1116 G>C (p.Gln372His) transcripts among the total mRNA pool. In contrast, family C shows evidence for maternal allele bias, with a lower peak height of the paternal c.464A relative to maternal c.464G variant (red asterisks). Collective data (D, right and E, right) suggest that the paternal exon 4-skipped transcripts are less abundant than the maternal c.464A>G (p.Asn155Ser) among the total mRNA pool.

Figure 2

Histopathological Findings in PYROXD1 Myopathy (A) Histopathological findings in skeletal muscle sections from family A (A-II1, quadriceps biopsy at 11 years of age), family B (B-II3, quadriceps biopsy at 16 years of age), and family C (C-II2, quadriceps biopsy at 4 years of age). Haemotoxylin and eosin (H&E) staining of muscle biopsy specimens from each family shows variation in fiber size, multiple internalized nuclei, and increased fibrous connective tissue. Immunofluorescent staining of skeletal muscle from A-II1 and C-II2 and immunoperoxidase staining from B-II3 demonstrate inclusions highly immunoreactive to desmin, myotillin, and alpha-actin (and αB-crystallin, not shown). H&E and immunoperoxidase images are provided via Hospital Pathology without a scale bar. Fibers in sequential sections of B-II3 are marked with a yellow star. (B) Electron microscopy of muscle biopsy specimens. Family A: (i) small atrophic fiber with a central nucleus and loss of sarcomeric organization; (ii) large region of Z-band streaming with only occasional areas of normal sarcomeric register; and (iii, iv) atrophic fibers showing total loss of sarcomeric register, loss of thick filaments, and prominent Z-bands sometimes forming small nemaline bodies. Family B: (v) Large fibers show large central minicore-like regions devoid of normal myofibrillar structure and lacking mitochondria and organelles, with adjacent small fibers showing total loss of sarcomeric structure, accumulations of thin filaments, and loss of thick filaments; (vi) many large fibers have multiple internalized nuclei, often in clusters; and (vii) fibers show thin filament accumulations with electron-dense aggregates that resemble thickened z-lines and small nemaline bodies. Family C: (viii) Small atrophic fiber with loss of sarcomeric register. A large fiber shows multiple areas of Z-band streaming and a minicore-like region with absence of normal myofibrillar structure.

Figure 3

PYROXD1 Is an Oxidoreductase (A) A schematic of PYROXD1 with functional domains and identified missense variants created using DOG 2.0. Family A, Δexon3 and p.Gln372His (Q372H, green); families B and D, p.Asn155Ser (N155S); family C, Δexon4 and p.Asn155Ser (N155S, red). (B) PYROXD1 homology model derived from eight homologous crystal structures (see Subjects and Methods). A co-ordinated FAD co-factor and the position of each identified variant on the crystal structure of PYROXD1 are highlighted in green (family A, Δexon3 and Q372H) and pink (family C: Δexon4 and N155S) as in (A). (C) The identified missense variants p.Asn155Ser and p.Gln372His are evolutionarily conserved to primitive eukaryotes (Uniprot identifiers): human (Q8WU10), bovine (A7YVH9), chicken (F1NPI8), Xenopus (B1WAU8), Danio rerio (Q6PBT5), Dictyostelium (Q54H36). (D) Living wild-type (WT, BY4742) yeast cells expressing human PYROXD1-GFP, PYROXD1-N155S-GFP, or PYROXD1-Q372H-GFP were observed by fluorescence microscopy with GFP filters and DIC optics. The merge represents the merge between the GFP and DIC images. (E) Cultures of non-transformed wild-type (WT) or glr1Δ yeast, or glr1Δ yeast transformed with expression plasmids bearing wild-type, N155S-, or Q372H-PYROXD1 were spotted at the indicated concentration (OD_600nm) on rich (YPD) or on solid medium containing 3 mM H₂O₂. Plates were incubated at 30°C and observed after 48 hr. (F) Western blot of non-transformed wild-type (WT) and glr1Δ, as well as glr1Δ yeast transformed with PYROXD1 expression vectors. The black arrow indicates PYROXD1 and the lower panel shows the protein-stained membrane used as loading control.

Figure 4

Affected Individuals with PYROXD1 Variants Show Reduced or Near-Normal Levels of PYROXD1 (A) Western blot of skin fibroblasts from two controls (42 and 46 years of age) and affected siblings from family A (29 and 26 years) and family C (6 and 8 years). HEK293 cells transfected with a plasmid encoding human PYROXD1 establishes the apparent molecular weight of PYROXD1 at ∼60 kDa (UT, untransfected; T, transfected). A non-specific (NS) band is indicated by an arrow. Levels of PYROXD1 are reduced in family A but not different to control levels in family C. (B) Western blot of A-II1 triceps (11 years) (Abcam cat# ab122458; RRID: AB_11129858) shows reduced levels of PYROXD1 relative to three age-matched control biopsy specimens (quadriceps 11 years, 10 years, 15 years). Loading controls: β-tubulin and GAPDH control for overall protein content, with one cytoskeletal and one cytoplasmic marker; emerin controls for the number of nuclei; skeletal α-actinin controls for myofibrillar content. (C) Western blot of myoblasts extracts from control subject and B-II3. Extracts from COS-1 cells transfected with the 500 aa human PYROXD1 cDNA (GenBank: NM_024854.3) was used as size control.

Figure 5

PYROXD1 Shows Both Nuclear and Striated Immunolocalization in Human Muscle (A) Control stretched human skeletal (quadriceps) muscle co-stained with α-PYROXD1 (red), myotilin or desmin (green), and DAPI (blue). PYROXD1 intensely labels peripheral myonuclei, as well as showing two different patterns of striated labeling. Right: In this fiber, PYROXD1 brightly labels consecutive myonuclei and shows cytoplasmic striated labeling that aligns with desmin (Z-band region) and interdigitates between Z-bands (M-line region). Left: PYROXD1 does not brightly label myonuclei (asterisks show the position of DAPI-labeled nuclei) and shows a broader banding of striated labeling spanning the breadth of the I-band. (B) Control and AII-1 tricep cross-sectioned human skeletal muscle co-stained with α-PYROXD1 (red), α-desmin (green), and DAPI (blue). The triceps muscle from AII-1 bears large inclusions that positively label for desmin, split fibers, and multiple internalized nuclei that brightly label for PYROXD1. Coverslips were imaged on a Leica SP5 confocal and single Z-planes are presented.

Figure 6

PYROXD1 Shows Nuclear Localization in Human Skin Fibroblasts and Transfected Cos-7 Cells (A) Untransfected Cos-7 cells (top row) and Cos-7 cells transfected with human wild-type, p.Asn155Ser (N155S)-, or p.Gln372His (Q372H)-PYROXD1 expression constructs co-stained for PYROXD1 (green) and lamin A/C (red) shows enriched labeling for PYROXD1 within the nucleus, as well as labeling of cytoplasmic networks. For transfected Cos-7 cells (bottom three rows), asterisks demark untransfected cells within the field, with much lower levels of PyroxD1, that show very weak labeling under optimum capture conditions for transfected cells. (B) Nuclear-cytoplasmic labeling is observed for endogenous PYROXD1 in primary human fibroblasts.

Figure 7

PYROXD1 Shows Nuclear-Cytoplasmic Localization in Zebrafish Myofibers, with Aggregates Induced by Expression of Asn155Ser and Gln372His Variants (A) In situ hybridization on 24 hpf zebrafish shows widespread ryroxd1 expression including the trunk musculature (M) in both lateral and dorsal views. (B–D) Immunolabeling of vibratome-sectioned embryos 4 days post fertilization (dpf) expressing eGFP-conjugated human PYROXD1, either wild-type (B), p.Asn155Ser (C; N155S), or p.Gln372His (D; Q372H) via a muscle-specific actin promoter (actc1b) together with the Z-disk marker Actinin3-mCherry or nuclear marker histone H2A-mCherry. PYROXD1 localizes to myonuclei and shows striated labeling of sarcomeres. Small regions of thickening and sarcomeric disruption (arrowheads and inset) were observed after heterologous expression of N155S- or Q372H-PYROXD1, but not with wild-type PYROXD1.

Figure 8

Zebrafish Deficient for RyroxD1 Show Sarcomeric Disorganization, Myofibrillar Aggregates, and a Defect in Swimming (A) Quantitative PCR for ryroxd1 mRNA levels in control (uninjected) and Ryroxd1 morpholino-injected (Ryroxd1 ex2 splice MO) zebrafish at 2 dpf. (B) Western blot for Ryroxd1 protein and α-tubulin loading control in control (uninjected) and Ryroxd1 morpholino-injected zebrafish (D = Ryroxd1 double morpholino, ATG = Ryroxd1 ATG morpholino, and splice = Ryroxd1 splice-site targeting morpholino) at 48 hpf demonstrates effective reduction of Ryroxd1. (C) Ryroxd1 ATG-single and ATG/splice double morphants show a significant reduction, of 48% and 73% respectively, in maximum acceleration in a touch-evoke response assay at 2 dpf compared to control zebrafish injected with a GFP targeting morpholino (Cont). (D and E) Antibody labeling of Ryroxd1 double morphants at 48 hpf (D) and 96 hpf (E) for Actinin2 and phalloidin show disruption of muscle structure compared to uninjected controls. (E) At 96 hpf, Ryroxd1 double morphants show severe disruption of the musculature with remnants of fragmented muscle fibers evident (arrows). For (A) and (C), error bars represent SEM for three independent replicate experiments comprising 15 fish in each, ^∗∗p < 0.01.

Figure 9

Human PYROXD1 Rescues Muscle Pathology and Swimming Defects in Ryroxd1 Morphants and EM of Zebrafish Muscle Pathology (A) Representative images depicting the range of severity of muscle defects in Ryroxd1 double morphants. No obvious phenotype (none), embryos displaying occasional broken fibers and actin accumulation (mild, arrows), severe fragmentation of muscle fibers (severe, arrowheads), severe loss of fiber integrity and accumulation of actin at the myosepta (severe, arrows). (B) Quantification of phenotypes (as in A) observed in Ryroxd1 double morphants (Ryroxd1 D MO) injected with either 0.5 ng/μL or 1.0 ng/μL wild-type (wt) wtPYROXD1-eGFP RNA compared to Ryroxd1 double morpholino injection alone (no RNA). 15–20 animals were scored per condition, ^∗p < 0.05. (C) Western blot for GFP and α-tubulin in GFP morpholino-injected control embryos (cont), Ryroxd1 double morpholino-injected zebrafish (no RNA), and Ryroxd1 double morpholino-injected zebrafish co-injected with either 0.5 ng/μL or 1.0 ng/μL wtPYROXD1-eGFP RNA, demonstrating translation of the injected mRNA. (D) Ryroxd1 double morphants injected with human wtPYROXD1-eGFP mRNA show dose-dependent rescue in a touch-evoke response assay at 2 dpf. Ryroxd1 double morphants injected with 1.0 ng/μL human wtPYROXD1-eGFP show a 61% increase in maximum acceleration compared to Ryroxd1 double morphants (no RNA), achieving similar levels of maximum acceleration to wild-type embryos injected with a control GFP targeting morpholino (GFP MO). (E) Electron micrograph of muscle (i) in a wild-type uninjected 96 hpf zebrafish embryo; examination of Ryroxd1 double morphant embryos (ii–iv) demonstrates myofibrillar fragmentation (ii, arrows) with mitochondrial infiltration, small nemaline-like bodies (iii, black arrowheads), and Z-disk fragmentation and loss (iii–iv; black arrowheads). Scale bar represents 1 μm. Error bars represent SEM for three independent replicate experiments comprising 15 animals in each replicate experiment, ^∗p < 0.05, ^∗∗p < 0.01.