Article Figures & Data
Figures
- Figure 1 Family pedigrees, muscle imaging, and genetic investigation
(A) Family pedigrees are shown. Filled-in symbols indicate individuals with muscle weakness. Empty symbols indicate unaffected individuals without any medical history or related complaint of muscle weakness, dementia, or bone disease. Asterisks indicate individuals whose DNA was used for this study. In family 1, DNA was used for whole-exome sequencing and segregating study. In family 2, it was used for Sanger sequencing. Arrows and arrowheads indicate individuals who underwent clinical examination and muscle biopsy, respectively. (B) Muscle CT was evaluated 8 years after onset in patient III-1 and 10 years after onset in the other patients. The images show slices of the trunk (first row), proximal arms (second row), and proximal (third row) and distal legs (fourth row). At the level of the trunk, all of the patients exhibited moderate-to-severe atrophy in the paraspinal (P) muscles. At the level of the proximal arms, the biceps brachii (BB) was commonly affected, although the triceps brachii (TB) and brachioradialis (BR) were spared. At the level of the proximal legs, the biceps femoris (BF), semimembranosus (SM), adductor magnus (AM), and vastus intermedius (VI) were predominantly affected. Less predominant atrophy was observed in the sartorius (SA), semitendinosus (ST), vastus lateralis (VL), and vastus medialis (VM) muscles. The atrophy of the semitendinosus muscle of patient IV-1 appeared asymmetrical. The rectus femoris (RF) and gracilis (G) muscles were relatively spared in patients III-1 and III-2, whereas they appeared to be affected later in patients IV-1 and IV-2. At the level of the distal legs, the soleus (S) was severely affected in all patients. In contrast, the peroneus longus (PL) gastrocnemius (GC), and tibialis posterior (TP) muscles were relatively spared. The tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were affected to varying degrees in each individual. (C) The sequence of the identified hnRNPA1 mutation and its conservation among species are shown. Sanger sequencing confirmed the heterozygous G to A substitution (indicated by arrows) at the position chr12: 54,677,628, which corresponds to c. 940G>A in exon 9 (NM_031157). The substitution leads to p.D314N (NP_112420), and this amino acid is conserved among species.
- Figure 2 Muscle histopathology
Hematoxylin & eosin (A, B), modified Gomori trichrome (C, D), Nicotinamide adenine dinucleotide–tetrazolium reductase (E, F), cytochrome c oxidase (COX) (G, H), serial, and ATPase pH4.5 (I, J) staining of the biopsied muscle samples from the biceps brachii of patient III-2 in family 1 (left panels, A, C, E, G, I) and the triceps brachii of patient IV-1 in family 2 (right panels, B, D, F, H, J) are shown. Hypertrophic fibers larger than 100 μm and angulated or rounded atrophic fibers are shown (A, B). Highly increased central nuclei and a fraction of fibers with pyknotic nuclear clumps are present (B). Rimmed vacuoles are located in atrophic fibers, which tend to make small groups (C, D). Disorganization of the myofibrillar network is observed (F). COX staining does not display complete COX-deficient myofibers (G, H). On ATPase pH4.5, the type distribution is almost equal, and atrophic fibers are observed in both type 1 and type 2 fibers (I, J). Type grouping is essentially negative except for the subtle finding of several type 1 fibers making a small group (I, J). Scale bars = 100 μm.
- Figure 3 Ultrastructural analysis of muscle
Ultrastructural findings of patient III-2 in family 1 are shown. The enclosed section of E is enlarged in F. The autophagic vacuoles containing myeloid bodies and glycogen granules are located among myofibrils (A, B), and neighboring myonuclei (C–F). Myonuclei (indicated by N) located in subsarcolemma (C) or sarcoplasm (D) are irregularly shaped and have indentation (an arrow). Scale bars are embedded in each panel.
- Figure 4 Multiple immunofluorescence for hnRNPA1 and related proteins
Representative microphotographs of transverse cryosections from the biopsied skeletal muscle of patient III-2 in family 1. (A–H) Aberrant subsarcolemmal/perinuclear aggregation (arrowheads) and increased sarcoplasmic retention of heterogeneous nuclear ribonucleoprotein (hnRNPA1) were mainly evident in atrophic fibers. Inset in H is a higher magnification of the boxed area (scale bar = 10 μm). The subsarcolemmal/perinuclear hnRNPA1 aggregates were often colocalized with ubiquitin (Ub, A–D, arrowheads) and SQSTM1/p62 (E–H, arrows). Note the close association of hnRNPA1/Ub double-positive aggregation with the rimmed vacuole (A–D, arrows). (I–L) In the atrophic fibers, transactive response DNA binding protein 43 kDa (TDP-43)/Ub double-positive aggregation (arrowheads) was also observed with the cytoplasmic mislocalization and nuclear depletion of TDP-43 (arrows). (M–P) The aberrant aggregation of hnRNPA1 was occasionally triple-labeled with phosphorylated TDP-43 and sequestome-1/p62 (SQSTM1/p62), closely adjacent to rimmed vacuoles (arrows). (Q–X) The rimmed vacuoles were often related to Ub, phosphorylated SQSTM1/p62 (Q–T, arrows), and hnRNPA2B1 (U–X, asterisks indicate a rimmed vacuole–carrying fiber). TO-PRO-3: nuclear staining (C, G, K, W). Scale bars = 50 μm.
- Figure 5 Multiple immunohistochemistry for various RNA-binding proteins and VCP/p97
Representative microphotographs of transverse cryosections from the specimens of patient III-2 in family 1. (A–D) Note the perinuclear and subsarcolemmal aggregation of fused in sarcoma/translated in liposarcoma (FUS/TLS) with sparse staining in cytoplasm (arrows), whereas FUS/TLS mislocalization was scarcely observed. (E–H) In atrophic fibers (asterisks), diffuse cytoplasmic expression and extranuclear/subsarcolemmal aggregation of TATA-binding protein–associated factor 2N (TAF15) were frequently found. The aggregation is partially colabeled with ubiquitin (arrowheads). (I–L) In addition to heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and A2B1, Ewing sarcoma breakpoint region 1 (EWSR1) and sequestome-1/p62 (SQSTM1/p62) double-positive aggregation was observed in rimmed vacuoles (arrows). (M–P) Note the multisystem proteinopathy 1–linked valosin-containing protein (VCP)/p97, SQSTM1/p62, and hnRNPA1 triple-labeled aggregates in the rimmed vacuoles (arrows). (Q–T) In atrophic fibers, amyotrophic lateral sclerosis/distal myopathy–linked matrin-3 was aberrantly involved in the subsarcolemmal SQSTM1/p62-positive aggregates (arrowheads). Scale bars = 50 μm.
Tables
Data Supplement
Files in this Data Supplement:
- Figure e-1 - PDF
- e-Methods - Microsoft Word file
- Table e-1 - Microsoft Word file
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