TY - JOUR
AU1 - Bouman, Karlijn
AU2 - Küsters, Benno
AU3 - De Winter, Josine M
AU4 - Gillet, Cynthia
AU5 - Van Kleef, Esmee S B
AU6 - Eshuis, Lilian
AU7 - Brochier, Guy
AU8 - Madelaine, Angeline
AU9 - Labasse, Clémence
AU1 - Boulogne, Claire
AU1 - Van Engelen, Baziel G M
AU1 - Ottenheijm, Coen A C
AU1 - Romero, Norma B
AU1 - Voermans, Nicol C
AU1 - Malfatti, Edoardo
AB - Abstract Nemaline myopathy type 6 (NEM6), KBTBD13-related congenital myopathy is caused by mutated KBTBD13 protein that interacts improperly with thin filaments/actin, provoking impaired muscle-relaxation kinetics. We describe muscle morphology in 18 Dutch NEM6 patients and correlate it with clinical phenotype and pathophysiological mechanisms. Rods were found in in 85% of biopsies by light microscopy, and 89% by electron microscopy. A peculiar ring disposition of rods resulting in ring-rods fiber was observed. Cores were found in 79% of NEM6 biopsies by light microscopy, and 83% by electron microscopy. Electron microscopy also disclosed granulofilamentous protein material in 9 biopsies. Fiber type 1 predominance and prominent nuclear internalization were found. Rods were immunoreactive for α-actinin and myotilin. Areas surrounding the rods showed titin overexpression suggesting derangement of the surrounding sarcomeres. NEM6 myopathology hallmarks are prominent cores, rods including ring-rods fibers, nuclear clumps, and granulofilamentous protein material. This material might represent the histopathologic epiphenomenon of altered interaction between mutated KBTBD13 protein and thin filaments. We claim to classify KBTBD13-related congenital myopathy as rod-core myopathy. Congenital nemaline myopathy type 6 (NEM6), Cores, Electron microscopy, Granulofilamentous protein material, KBTBD13, Myopathology, Nuclear clumps, Rods INTRODUCTION Nemaline myopathy (NEM) is one of the most common congenital myopathies with an estimated prevalence of 1 in 50.000 births (1). It is clinically diverse but usually manifests with hypotonia, muscle weakness, and skeletal deformities. So far, 14 genes are known to be implicated in NEM: α-actin 1 (ACTA1), α- and β-tropomyosin (TPM3 and TPM2), nebulin (NEB), leiomodin-3 (LMOD3), troponin T (TNNT1 and TNNT3), cofilin 2 (CFL2), unconventional myosin 18B (MYO18B), myopalladin (MYPN), Ryanodine receptor type 3 (RYR3), kelch family members 40 (KLHL40) and 41 (KLHL41), and kelch repeat and BTB (POZ) Domain Containing 13 (KBTBD13) (2–11). NEM6 was first described in (Australian-)Dutch families and is characterized by peculiar slowness in movement, length-dependent weakness, and symmetric plus slowly progressive neck flexor and proximal limb muscle weakness (12–14). Symptoms start in childhood but many patients do not report until adolescence or adulthood. The peculiar slowness in movement, which is caused by slow muscle relaxation, precludes NEM6 patients to perform fast movements and contributes to falling when tripping. Slow muscle relaxation might be caused by structural changes in the thin filament (15). An autosomal dominant missense mutation in the KBTBD13 gene is found to be responsible for NEM6. Previous studies on the morphological features of 8 patients revealed the presence of numerous nemaline bodies, prominent myofibrillar disorganization resulting in core-like formations, type 1 fiber predominance and hypertrophy, and type 2 fiber atrophy (12, 16, 17). By using immunofluorescence, aggregates of Z-disk proteins including α-actinin, myotilin, and filamin-C were only observed in muscle biopsies from 2 patients (16). Electron microscopy showed variable numbers of nemaline bodies emerging from the Z-line and severe loss of myofibrillar organization. Additionally, foci of myofibrillar dissolution and destructive Z-disk filamentous masses containing a few rod-like bodies were seen (16). A recent study on the pathophysiological mechanisms of NEM6 revealed that impaired muscle-relaxation kinetics is caused by structural changes in the thin filament. KBTBD13 binds to actin—a major constituent of the thin filament—and KBTBD13-mutated proteins stiffen the thin filament and might thereby impair muscle-relaxation kinetics (15). Here we report on an extensive myopathological phenotyping study of 18 Dutch NEM6 patients that allowed us to reassess the histopathologic spectrum of NEM6-related congenital myopathy. MATERIALS AND METHODS Muscle biopsy specimen (m. quadriceps) of 18 Dutch NEM6 patients (≥16 years old) were included (male = 5; female = 13; mean age at biopsy: 43.2 ± 15.0 SD years). Diagnosis was based either on genetic evaluation (whole exome sequencing) or on typical clinical and histological alterations combined with genetic confirmation in a close relative. All patients gave informed consent and this study was conducted in accordance with the Declaration of Helsinki. We had stained slides from regular diagnostics for 15 NEM6 patients (male = 4, female = 11; mean age at biopsy: 40.6 ± 14.7 SD years). Frozen muscle biopsies were available for 5 NEM6 patients (male = 1, female = 4; mean age at biopsy: 31 ± 9.0 SD years). Further, muscle biopsies of 6 patients (m. quadriceps (n = 2); m. deltoid (n = 4), male = 3; female = 3; mean age at biopsy: 35 ± 14 SD) without specific histopathologic alteration were included to assess the specificity of our findings. To add consistency to the study, we only used the 2 control quadriceps muscles to compare to the quadriceps muscles of all the NEM6 patients for all data related to fiber type distribution. Material suitable for electron microscopy was available for all 18 NEM6 patients and for 2 patients without specific ultrastructural alterations. Clinical Phenotyping The clinical characteristics of our cohort were retrospectively analyzed by evaluating medical records. All patients had undergone a medical history and physical examination by a neurologist specialized in neuromuscular disorders at the time of the muscle biopsy and/or at the most recent visit. Recent clinical data of 8 NEM6 patients were available thanks to an ongoing clinical study on nemaline myopathies at the Radboud University Medical Center. Patients had been referred to a cardiologist, pulmonologist, or to other specialists (physiotherapist, physiatrist) on indication. We used a binary scoring system to describe the presence (+) or absence (−) of exercise intolerance, cardiac involvement, and muscle slowness. General muscle strength and quadriceps muscle strength according to the Medical Research Council (MRC) grading scale were taken from general neurologic examination that was performed in regular diagnostics, during follow-up and/or in our ongoing clinical study on nemaline myopathies. Muscle strength was classified as absent (−; no muscle weakness, all muscles MRC5), mild (+; mild muscle weakness, all muscle 4 ≤ MRC < 5), moderate (++; moderate muscle weakness, all muscles 3 ≤ MRC < 4) and severe (+++; severe muscle weakness, at least one muscle group MRC2). Quadriceps muscle strength was classified by a parallel grading scale. Histochemistry and Histoenzymology Muscle biopsy samples were frozen in liquid nitrogen-cooled isopentane and cut (8 μm) using Leica CM3050S cryostat at −23°C. Muscle biopsy specimens were processed in regular diagnostics for routine histological reactions (hematoxylin and phloxine (HPhlox) or hematoxylin and eosin (H&E), modified Gömöri trichrome (mGT), reduced nicotinamide adenosine dinucleotide tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), cytochrome C oxidase (COX), cytochrome C oxidase—succinate dehydrogenase (COX-SDH) and adenosine triphosphatase (ATPase) 4.2 according standardized methods (18). Fifteen slides for H&E/HPhlox, 13 slides for mGT, 14 slides for NADH, and 13 slides for ATPase 4.2 were available in our cohort of NEM6 patients. In one case (case 16), due to low quality of the available ATPase 4.2 slides, we performed fast myosin immunohistochemistry to corroborate the fiber-type distribution findings. Biopsies were classified as mildly, moderately, or severely affected by grading scales that are in accordance with diagnostic guidelines provided by the Radboud University Medical Center (Table 1). TABLE 1. Grading Scale of Pathologic Features in Muscle Biopsies (Percentage of Fibers Affected). . Normal . Mild . Moderate . Severe . HE/HPhlox  Internalized nuclei <3% 3–8% 8–30% >30%  Nuclear clumps Absent 0–20% >20%  Fiber regeneration <2% 2–10% >10%  Fiber splitting Absent 0–10% >10% mGT  Rods Absent 0–20% 20–40% >40% NADH  Cores Absent 0–20% 20–40% >40% ATPase 4.2/fast myosin  Fiber type 1 (%) 35–50% 50–70% 70–90% >90%  Fiber type 1 (dm) 40–80 µm 30–90 µm 20–100 µm <20 or >100  Fiber type 2 (dm) 40–80 µm 30–90 µm 20–100 µm <20 or >100 . Normal . Mild . Moderate . Severe . HE/HPhlox  Internalized nuclei <3% 3–8% 8–30% >30%  Nuclear clumps Absent 0–20% >20%  Fiber regeneration <2% 2–10% >10%  Fiber splitting Absent 0–10% >10% mGT  Rods Absent 0–20% 20–40% >40% NADH  Cores Absent 0–20% 20–40% >40% ATPase 4.2/fast myosin  Fiber type 1 (%) 35–50% 50–70% 70–90% >90%  Fiber type 1 (dm) 40–80 µm 30–90 µm 20–100 µm <20 or >100  Fiber type 2 (dm) 40–80 µm 30–90 µm 20–100 µm <20 or >100 Dm = diameter; µm = micrometer; % = percentage. Open in new tab TABLE 1. Grading Scale of Pathologic Features in Muscle Biopsies (Percentage of Fibers Affected). . Normal . Mild . Moderate . Severe . HE/HPhlox  Internalized nuclei <3% 3–8% 8–30% >30%  Nuclear clumps Absent 0–20% >20%  Fiber regeneration <2% 2–10% >10%  Fiber splitting Absent 0–10% >10% mGT  Rods Absent 0–20% 20–40% >40% NADH  Cores Absent 0–20% 20–40% >40% ATPase 4.2/fast myosin  Fiber type 1 (%) 35–50% 50–70% 70–90% >90%  Fiber type 1 (dm) 40–80 µm 30–90 µm 20–100 µm <20 or >100  Fiber type 2 (dm) 40–80 µm 30–90 µm 20–100 µm <20 or >100 . Normal . Mild . Moderate . Severe . HE/HPhlox  Internalized nuclei <3% 3–8% 8–30% >30%  Nuclear clumps Absent 0–20% >20%  Fiber regeneration <2% 2–10% >10%  Fiber splitting Absent 0–10% >10% mGT  Rods Absent 0–20% 20–40% >40% NADH  Cores Absent 0–20% 20–40% >40% ATPase 4.2/fast myosin  Fiber type 1 (%) 35–50% 50–70% 70–90% >90%  Fiber type 1 (dm) 40–80 µm 30–90 µm 20–100 µm <20 or >100  Fiber type 2 (dm) 40–80 µm 30–90 µm 20–100 µm <20 or >100 Dm = diameter; µm = micrometer; % = percentage. Open in new tab Immunohistochemistry Slides were processed for desmin (1:500; Clone D33, Code Nr. M 0760, Dako, Santa Clara, CA), αB-crystallin (1:2000; NCL-ABCrys-512, Leica Biosystems, Wetzlar, Germany) and fast myosin (1:200; NCL-MHCf, clone WB-MHCf, Leica Biosystems) by Leica Bond Max III using BOND Polymer Refine Detection kit (DS9800, Leica Biosystems) containing peroxide block (3%–4% hydrogen peroxide). For 5 patients, we had slides available that were previously stained in routine diagnostics against fast, slow, neonatal and developmental myosin. Slides were processed for α-actinin II (1:500; SIGMA A7811; Sigma Aldrich, St. Louis, MO), myotilin (1:100; NCL-myotilin, RSO34, Leica Biosystems) and titin (M10-1× (1:50; SC1492; GenScript Biotech, Piscataway, NJ) and N2A (1:100; T5650; US Biological, Salem, MA) using standardized protocols for immunofluorescence. All slides were incubated with secondary antibody AF555 (1:500; A21424; Alexa Fluor, ThermoFisher Scientific, Waltham, MA) or AF488 (1:500; A11034; Alexa Fluor, ThermoFisher Scientific). Slides processed for routine histological reactions and immunoperoxidase were scanned through the Aperio AT2 (Leica Biosystems) scanner connected to Aperio ImageScope (v12.4.0.5043) software. All pictures of slides stained by immunofluorescence were taken using a Zeiss microscope Imager.Z1 with an AxioCam MRc camera and AxioVs40 v.4.8.2.0 (2012) software connected to it. Confocal images were taken on Leica TCS SP8 × Confocal microscope using Leica Application Suite X 3.7.0.20979 (2019) software. Electron Microscopy Muscle specimens were fixed in 2% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.4), post fixed in 1% osmium tetroxide in Palade buffer (pH 7.4) with 0.25% potassium ferrocyanide-trihydrate, dehydrated in ethanol and propylene oxide, and embedded in Epon. Longitudinal and transverse ultrathin sections (80 nm) were cut on the UC6 Leica Ultramicrotome. Sections were double contrasted with uranyl acetate 2% for 15 minutes and Reynolds lead citrate for 6 minutes (19). The grids were observed using a Philips CM120 electron microscope (80 kV; Philips Electronic NV, Eindhoven, The Netherlands) connected to the Morada camera (Soft Imaging System) or using the JEOL JEM-1400 electron microscope (80 kV or 120 kV) connected to the Orius SC1000 CCD Camera or the Rio US1000 Camera (Gatan, Inc., Warrendale, PA). Biostatistical Analyses Statistical analysis was performed using IBM SPSS Statistics (version 25) for Windows. Descriptive statistics were used to describe histopathological and clinical characteristics of NEM6 patients and healthy controls. Pearson’s correlation coefficient was used to assess the clinic-morphological correlation. RESULTS Clinical and Genetic Features A systematic overview on clinical information is reported in Table 2. NEM6 patients suffered from slowly progressive proximal muscle weakness starting in childhood. A key characteristic included prominent weakness of the neck flexor muscles. Muscle weakness resulted in difficulties in running, jumping, and climbing stairs. Some patients experienced difficulties in walking resulting in intermittent use of walking aids. Manual muscle tests showed objective muscle weakness in all patients. General muscle weakness at most recent visit was mild in 5 patients, moderate in 4, and severe in 7 patients. Its severity was mostly determined by the severe neck flexor muscles, resulting in a substantial number of patients classified as severely affected, while all remained ambulatory. Quadriceps muscle weakness was absent in 10 patients, mild in 5, and moderate in 1. The neurological examination performed closest to the muscle biopsy disclosed 4 mildly affected, 8, and 4 severely affected patients. In contrast, 6 patients showed no quadriceps weakness, 6 had mild, and 3 had moderate weakness. None of our patients exhibited a dropped head. Muscle slowness was reported in 12 patients. There was no clinical myotonia, nor spontaneous muscle fiber discharge on electromyography. Six patients suffered from cardiac involvement. Left ventricle ejection fraction was decreased in 2 patients (40%–45% and 49%). Further, cardiac arrhythmias were seen in 3 patients, resulting in the implementation of an implantable cardioverter-defibrillator in 2 patients. One patient was diagnosed with an aneurysm of the atrial septum (Table 2). There was no respiratory involvement. TABLE 2. Clinical Features of NEM6 Patients. Patient . Gender . Age atonset symptoms . Current age (years) . Age at biopsy . General muscle weakness (age, years) closest tomoment of biopsy . Quadriceps muscle weakness (age, years) closest to moment of biopsy . Most recent general muscle weakness (age, years) . Most recentquadriceps muscle weakness(age, years) . Exercise intoler-ance . Muscle slowness . Cardiac abnormalities . Family . Genetic mutation . 1 F 10s Died (56 y/o,mamma ca) 34 ++ (35 y/o) ++ (35 y/o) ++ (37 y/o) + (37 y/o) + ? ? 1 c.1222C>T, p. Arg408Cys 2 F Childhood 61 38 ++ (38 y/o) ++ (38 y/o) +++ (56 y/o) ++ (56 y/o) + + + (LBBB, LVEF 40–45%) 2 c.1222C>T, p. Arg408Cys 3 F Childhood 78 57 +++ (57 y/o) − (57 y/o) +++ (76 y/o) − (76 y/o) − + − None c.1222C>A p. Arg408Ser 4 M ? Died(72 y/o) 67 ? (67 y/o) ? ? ? ? + − 2 c.1222C>T, p. Arg408Cys 5 F Childhood Died(45 y/o,colon ca) 34 ? (34 y/o) ? ? ? + ? − 2 c.1222C>T, p. Arg408Cys 6 M ? Died(76 y/o) 63 + (63 y/o) + (63 y/o) + (63 y/o) + (63 y/o) ? − ? 2 c.1222C>T, p. Arg408Cys 7 F ? 63 46 ++ (62 y/o) ++ (62 y/o) ++ (62 y/o) − (62 y/o) ? ? + (ventricular extrasystoles; trigeminy) 2 c.1222C>T, p. Arg408Cys 8 M Childhood Died(75 y/o) 68 +++ (64 y/o) + (64 y/o) +++ (64 y/o) + (64 y/o) + ? ? 2 c.1222C>T, p. Arg408Cys 9 F 10s 61 46 + (53 y/o) + (57 y/o) + (61 y/o) + (61 y/o) + + + (LVEF 49%) 1 c.1222C>T, p. Arg408Cys 10 F Childhood 31 16 + (16 y/o) + (23 y/o) +++ (30 y/o) − (30 y/o) + + + (aneurysm of atrial septum) 1 c.1222C>T, p. Arg408Cys 11 F Childhood 29 19 +++ (26 y/o) − (26 y/o) +++ (28 y/o) − (28 y/o) + ? − 1 c.1222C>T, p. Arg408Cys 12 F Childhood 52 42 ++ (45 y/o) + (50 y/o) +++ (51 y/o) − (51 y/o) + + + (ventricular tachycardia; ICD) 1 c.1222C>T, p. Arg408Cys 13 F Childhood 51 41 ++ (43 y/o) + (43 y/o) ++ (43 y/o) + (43 y/o) − + − None c.1170G>C, p. Lys390Asn 14 M Childhood 39 31 + (36 y/o) ? + (39 y/o) − (39 y/o) − + ? 1 c.1222C>T, p. Arg408Cys 15 F Childhood 36 28 +++ (29 y/o) − (29 y/o) + (36 y/o) − (36 y/o) + + − 1 c.1222C>T, p. Arg408Cys 16 F 30s 51 44 ++ (51 y/o) − (51 y/o): ++ (51 y/o) − (51 y/o) − + + (cardiac arrythmia; ICD) 1 c.1222C>T, p. Arg408Cys 17 M 40s 56 50 ++ (51 y/o) − (51 y/o) + (55 y/o) − (51 y/o) ? + − None c.742C>T p.(Arg248Cys) 18 F Infancy 36 33 ++ (33 y/o) − (33 y/o) +++ (35 y/o) − (35 y/o) − + − 1 c.1222C>T, p. Arg408Cys Patient . Gender . Age atonset symptoms . Current age (years) . Age at biopsy . General muscle weakness (age, years) closest tomoment of biopsy . Quadriceps muscle weakness (age, years) closest to moment of biopsy . Most recent general muscle weakness (age, years) . Most recentquadriceps muscle weakness(age, years) . Exercise intoler-ance . Muscle slowness . Cardiac abnormalities . Family . Genetic mutation . 1 F 10s Died (56 y/o,mamma ca) 34 ++ (35 y/o) ++ (35 y/o) ++ (37 y/o) + (37 y/o) + ? ? 1 c.1222C>T, p. Arg408Cys 2 F Childhood 61 38 ++ (38 y/o) ++ (38 y/o) +++ (56 y/o) ++ (56 y/o) + + + (LBBB, LVEF 40–45%) 2 c.1222C>T, p. Arg408Cys 3 F Childhood 78 57 +++ (57 y/o) − (57 y/o) +++ (76 y/o) − (76 y/o) − + − None c.1222C>A p. Arg408Ser 4 M ? Died(72 y/o) 67 ? (67 y/o) ? ? ? ? + − 2 c.1222C>T, p. Arg408Cys 5 F Childhood Died(45 y/o,colon ca) 34 ? (34 y/o) ? ? ? + ? − 2 c.1222C>T, p. Arg408Cys 6 M ? Died(76 y/o) 63 + (63 y/o) + (63 y/o) + (63 y/o) + (63 y/o) ? − ? 2 c.1222C>T, p. Arg408Cys 7 F ? 63 46 ++ (62 y/o) ++ (62 y/o) ++ (62 y/o) − (62 y/o) ? ? + (ventricular extrasystoles; trigeminy) 2 c.1222C>T, p. Arg408Cys 8 M Childhood Died(75 y/o) 68 +++ (64 y/o) + (64 y/o) +++ (64 y/o) + (64 y/o) + ? ? 2 c.1222C>T, p. Arg408Cys 9 F 10s 61 46 + (53 y/o) + (57 y/o) + (61 y/o) + (61 y/o) + + + (LVEF 49%) 1 c.1222C>T, p. Arg408Cys 10 F Childhood 31 16 + (16 y/o) + (23 y/o) +++ (30 y/o) − (30 y/o) + + + (aneurysm of atrial septum) 1 c.1222C>T, p. Arg408Cys 11 F Childhood 29 19 +++ (26 y/o) − (26 y/o) +++ (28 y/o) − (28 y/o) + ? − 1 c.1222C>T, p. Arg408Cys 12 F Childhood 52 42 ++ (45 y/o) + (50 y/o) +++ (51 y/o) − (51 y/o) + + + (ventricular tachycardia; ICD) 1 c.1222C>T, p. Arg408Cys 13 F Childhood 51 41 ++ (43 y/o) + (43 y/o) ++ (43 y/o) + (43 y/o) − + − None c.1170G>C, p. Lys390Asn 14 M Childhood 39 31 + (36 y/o) ? + (39 y/o) − (39 y/o) − + ? 1 c.1222C>T, p. Arg408Cys 15 F Childhood 36 28 +++ (29 y/o) − (29 y/o) + (36 y/o) − (36 y/o) + + − 1 c.1222C>T, p. Arg408Cys 16 F 30s 51 44 ++ (51 y/o) − (51 y/o): ++ (51 y/o) − (51 y/o) − + + (cardiac arrythmia; ICD) 1 c.1222C>T, p. Arg408Cys 17 M 40s 56 50 ++ (51 y/o) − (51 y/o) + (55 y/o) − (51 y/o) ? + − None c.742C>T p.(Arg248Cys) 18 F Infancy 36 33 ++ (33 y/o) − (33 y/o) +++ (35 y/o) − (35 y/o) − + − 1 c.1222C>T, p. Arg408Cys M = male; F = female; ca = carcinoma; LBBB = left bundle branch block; LVEF = left ventricular ejection fraction; ICD = Implantable cardioverter–defibrillator; muscle weakness: − = no muscle weakness (all muscles MRC 5); + = mild muscle weakness (all muscles MRC ≥ 4 but not MRC 5); ++ = moderate muscle weakness (all muscle MRC ≥ 3, but not MRC ≥ 4); +++ = severe muscle weakness (at least one muscle group MRC 2); ? = unknown; exercise intolerance, muscle slowness and cardiac abnormalities: − = absent; + = present; ? = unknown; boldface is used for indicating clinical data that were recently obtained in our ongoing clinical study at the Radboudumc. Open in new tab TABLE 2. Clinical Features of NEM6 Patients. Patient . Gender . Age atonset symptoms . Current age (years) . Age at biopsy . General muscle weakness (age, years) closest tomoment of biopsy . Quadriceps muscle weakness (age, years) closest to moment of biopsy . Most recent general muscle weakness (age, years) . Most recentquadriceps muscle weakness(age, years) . Exercise intoler-ance . Muscle slowness . Cardiac abnormalities . Family . Genetic mutation . 1 F 10s Died (56 y/o,mamma ca) 34 ++ (35 y/o) ++ (35 y/o) ++ (37 y/o) + (37 y/o) + ? ? 1 c.1222C>T, p. Arg408Cys 2 F Childhood 61 38 ++ (38 y/o) ++ (38 y/o) +++ (56 y/o) ++ (56 y/o) + + + (LBBB, LVEF 40–45%) 2 c.1222C>T, p. Arg408Cys 3 F Childhood 78 57 +++ (57 y/o) − (57 y/o) +++ (76 y/o) − (76 y/o) − + − None c.1222C>A p. Arg408Ser 4 M ? Died(72 y/o) 67 ? (67 y/o) ? ? ? ? + − 2 c.1222C>T, p. Arg408Cys 5 F Childhood Died(45 y/o,colon ca) 34 ? (34 y/o) ? ? ? + ? − 2 c.1222C>T, p. Arg408Cys 6 M ? Died(76 y/o) 63 + (63 y/o) + (63 y/o) + (63 y/o) + (63 y/o) ? − ? 2 c.1222C>T, p. Arg408Cys 7 F ? 63 46 ++ (62 y/o) ++ (62 y/o) ++ (62 y/o) − (62 y/o) ? ? + (ventricular extrasystoles; trigeminy) 2 c.1222C>T, p. Arg408Cys 8 M Childhood Died(75 y/o) 68 +++ (64 y/o) + (64 y/o) +++ (64 y/o) + (64 y/o) + ? ? 2 c.1222C>T, p. Arg408Cys 9 F 10s 61 46 + (53 y/o) + (57 y/o) + (61 y/o) + (61 y/o) + + + (LVEF 49%) 1 c.1222C>T, p. Arg408Cys 10 F Childhood 31 16 + (16 y/o) + (23 y/o) +++ (30 y/o) − (30 y/o) + + + (aneurysm of atrial septum) 1 c.1222C>T, p. Arg408Cys 11 F Childhood 29 19 +++ (26 y/o) − (26 y/o) +++ (28 y/o) − (28 y/o) + ? − 1 c.1222C>T, p. Arg408Cys 12 F Childhood 52 42 ++ (45 y/o) + (50 y/o) +++ (51 y/o) − (51 y/o) + + + (ventricular tachycardia; ICD) 1 c.1222C>T, p. Arg408Cys 13 F Childhood 51 41 ++ (43 y/o) + (43 y/o) ++ (43 y/o) + (43 y/o) − + − None c.1170G>C, p. Lys390Asn 14 M Childhood 39 31 + (36 y/o) ? + (39 y/o) − (39 y/o) − + ? 1 c.1222C>T, p. Arg408Cys 15 F Childhood 36 28 +++ (29 y/o) − (29 y/o) + (36 y/o) − (36 y/o) + + − 1 c.1222C>T, p. Arg408Cys 16 F 30s 51 44 ++ (51 y/o) − (51 y/o): ++ (51 y/o) − (51 y/o) − + + (cardiac arrythmia; ICD) 1 c.1222C>T, p. Arg408Cys 17 M 40s 56 50 ++ (51 y/o) − (51 y/o) + (55 y/o) − (51 y/o) ? + − None c.742C>T p.(Arg248Cys) 18 F Infancy 36 33 ++ (33 y/o) − (33 y/o) +++ (35 y/o) − (35 y/o) − + − 1 c.1222C>T, p. Arg408Cys Patient . Gender . Age atonset symptoms . Current age (years) . Age at biopsy . General muscle weakness (age, years) closest tomoment of biopsy . Quadriceps muscle weakness (age, years) closest to moment of biopsy . Most recent general muscle weakness (age, years) . Most recentquadriceps muscle weakness(age, years) . Exercise intoler-ance . Muscle slowness . Cardiac abnormalities . Family . Genetic mutation . 1 F 10s Died (56 y/o,mamma ca) 34 ++ (35 y/o) ++ (35 y/o) ++ (37 y/o) + (37 y/o) + ? ? 1 c.1222C>T, p. Arg408Cys 2 F Childhood 61 38 ++ (38 y/o) ++ (38 y/o) +++ (56 y/o) ++ (56 y/o) + + + (LBBB, LVEF 40–45%) 2 c.1222C>T, p. Arg408Cys 3 F Childhood 78 57 +++ (57 y/o) − (57 y/o) +++ (76 y/o) − (76 y/o) − + − None c.1222C>A p. Arg408Ser 4 M ? Died(72 y/o) 67 ? (67 y/o) ? ? ? ? + − 2 c.1222C>T, p. Arg408Cys 5 F Childhood Died(45 y/o,colon ca) 34 ? (34 y/o) ? ? ? + ? − 2 c.1222C>T, p. Arg408Cys 6 M ? Died(76 y/o) 63 + (63 y/o) + (63 y/o) + (63 y/o) + (63 y/o) ? − ? 2 c.1222C>T, p. Arg408Cys 7 F ? 63 46 ++ (62 y/o) ++ (62 y/o) ++ (62 y/o) − (62 y/o) ? ? + (ventricular extrasystoles; trigeminy) 2 c.1222C>T, p. Arg408Cys 8 M Childhood Died(75 y/o) 68 +++ (64 y/o) + (64 y/o) +++ (64 y/o) + (64 y/o) + ? ? 2 c.1222C>T, p. Arg408Cys 9 F 10s 61 46 + (53 y/o) + (57 y/o) + (61 y/o) + (61 y/o) + + + (LVEF 49%) 1 c.1222C>T, p. Arg408Cys 10 F Childhood 31 16 + (16 y/o) + (23 y/o) +++ (30 y/o) − (30 y/o) + + + (aneurysm of atrial septum) 1 c.1222C>T, p. Arg408Cys 11 F Childhood 29 19 +++ (26 y/o) − (26 y/o) +++ (28 y/o) − (28 y/o) + ? − 1 c.1222C>T, p. Arg408Cys 12 F Childhood 52 42 ++ (45 y/o) + (50 y/o) +++ (51 y/o) − (51 y/o) + + + (ventricular tachycardia; ICD) 1 c.1222C>T, p. Arg408Cys 13 F Childhood 51 41 ++ (43 y/o) + (43 y/o) ++ (43 y/o) + (43 y/o) − + − None c.1170G>C, p. Lys390Asn 14 M Childhood 39 31 + (36 y/o) ? + (39 y/o) − (39 y/o) − + ? 1 c.1222C>T, p. Arg408Cys 15 F Childhood 36 28 +++ (29 y/o) − (29 y/o) + (36 y/o) − (36 y/o) + + − 1 c.1222C>T, p. Arg408Cys 16 F 30s 51 44 ++ (51 y/o) − (51 y/o): ++ (51 y/o) − (51 y/o) − + + (cardiac arrythmia; ICD) 1 c.1222C>T, p. Arg408Cys 17 M 40s 56 50 ++ (51 y/o) − (51 y/o) + (55 y/o) − (51 y/o) ? + − None c.742C>T p.(Arg248Cys) 18 F Infancy 36 33 ++ (33 y/o) − (33 y/o) +++ (35 y/o) − (35 y/o) − + − 1 c.1222C>T, p. Arg408Cys M = male; F = female; ca = carcinoma; LBBB = left bundle branch block; LVEF = left ventricular ejection fraction; ICD = Implantable cardioverter–defibrillator; muscle weakness: − = no muscle weakness (all muscles MRC 5); + = mild muscle weakness (all muscles MRC ≥ 4 but not MRC 5); ++ = moderate muscle weakness (all muscle MRC ≥ 3, but not MRC ≥ 4); +++ = severe muscle weakness (at least one muscle group MRC 2); ? = unknown; exercise intolerance, muscle slowness and cardiac abnormalities: − = absent; + = present; ? = unknown; boldface is used for indicating clinical data that were recently obtained in our ongoing clinical study at the Radboudumc. Open in new tab Genetics revealed that 15 patients harbored the Dutch KBTBD13 c.1222C>T p.(Arg408Cys) founder variant. Pedigree analysis of 6 generations showed that all patients with this founder mutation belong to either of 2 large families. Patient 3 harbored the c.1222C>A p.(Arg408Ser) variant. Patient 13 harbored the c.1170G>C, p.(Lys390Asn) variant and patient 17 harbored the c.742C>T p.(Arg248Cys) variant. The latter 3 patients are not related to each other nor to the other 2 families. Histochemistry and Histoenzymology Histologic grading scale and detailed pathologic findings are reported in Tables 1 and 3, respectively. The 6 control muscle biopsies showed no alterations. With H&E or HPhlox (n = 15) we observed nuclear clumps in 7.5% ± 2.6% (95% CI, 4.9–10) of fibers of 15 biopsies, showing overall severely atrophic fibers (Fig. 1A). No correlation was found between the number of nuclear clumps and the age at biopsy (Spearman’s correlation, p > 0.05). Internalized, often multiple, nuclei were seen in 41% ± 11% (95% CI, 30–51) of fibers (Fig. 1A). Fiber splitting was present in 7 biopsies (1.1% ± 0.53 [95% CI, 0.60–1.7] fibers) (Fig. 1B). There was regeneration in 2.7% ± 0.70% (95% CI, 2.0–3.4) of fibers (n = 11) (data not shown). mGT staining (n = 13) disclosed multiple, small, or confluent reddish inclusions corresponding to rods in 47% ± 15% (95% CI, 32–63) of fibers in 11 samples (Fig. 1C). Rods were found both in subsarcolemmal regions and/or sarcoplasm. Larger clusters were found preferentially centralized (Fig. 1C), while smaller clusters were randomly dispersed in the sarcoplasm. In 6 patients, sarcoplasmic rods organized in a star-shaped fashion were seen (Fig. 1C). Interestingly, there was a peculiar ring (circular) disposition of rods along the sarcolemma defined as ring-rods fibers in 5 biopsies (Fig. 1D). The ring-rods fibers were small and round, individually located or gathered in small groups (data not shown). Only 2 samples failed to show rods (Table 3). NADH-TR (n = 14) revealed cores in 27% ± 12% (95% CI, 15–39) of fibers from 11 biopsies (Table 3; Fig. 1E, F). The cores were single (n = 10), multiple (n = 1), round or oval, with variably defined borders, and different localization (central, eccentric, peripheral). In all but one sample expressing rods in mGT, there were cores in NADH-TR. All biopsies with cores showed the presence of rods. SDH and COX additionally showed areas without oxidative staining corresponding to cores, enabling us to conclude that mitochondria are absent. The areas occupied by rods or cores were devoid of ATPase reaction (data not shown). Some fibers showed intense subsarcolemmal NADH-TR, SDH, and COX histoenzymatic activity, suggesting a mitochondrial accumulation in subsarcolemmal regions, which was confirmed by electron microscopy (data not shown). These same areas appeared also fuchsinophilic at mGT staining, indicating the coexistence of rods and mitochondria. Occasionally, blue fibers were seen with COX-SDH, indicating COX deficiency (data not shown). FIGURE 1. Open in new tabDownload slide Morphologic features in NEM6 patients, light microscopy. (A) Nuclear clumps (arrow) and single or multiple internalized nuclei (open arrow) in most fibers (H&E). (B) Fiber splitting (arrow). Note the presence of internalized nuclei along the membrane invagination leading to fiber splitting (HPhlox). (C) Star-shaped rod cluster (arrow) and variably shaped fuchsinophilic inclusions corresponding to rods (open arrow) (mGT). (D) High magnification of subsarcolemmal disposition of reddish inclusions resulting in ring-rods fiber. (E) Core-like areas (arrow) (NADH-TR). (F) High magnification of a large sarcoplasmic core (arrow) (NADH-TR). FIGURE 1. Open in new tabDownload slide Morphologic features in NEM6 patients, light microscopy. (A) Nuclear clumps (arrow) and single or multiple internalized nuclei (open arrow) in most fibers (H&E). (B) Fiber splitting (arrow). Note the presence of internalized nuclei along the membrane invagination leading to fiber splitting (HPhlox). (C) Star-shaped rod cluster (arrow) and variably shaped fuchsinophilic inclusions corresponding to rods (open arrow) (mGT). (D) High magnification of subsarcolemmal disposition of reddish inclusions resulting in ring-rods fiber. (E) Core-like areas (arrow) (NADH-TR). (F) High magnification of a large sarcoplasmic core (arrow) (NADH-TR). TABLE 3. Histopathologic and Immunohistochemical Features of NEM6 and Controls. Disease . Routine histological reactions . Immunohistochemistry . . HE/HPhlox . mGT . NADH . ATPase 4.2/fast myosin . Immunoperoxidase . Immunofluorescence . Internalized nuclei (%) . Nuclear clumps (%) . Fiber splitting (%) . Fiber regenera-tion (%) . Rods (%) . Cores (%) . Fiber type 1 (%) . Fiber type 1 dm (um) . Minimal fiber type 1 dm (um) . Maximal fiber type 1 dm (um) . Fiber type 2 dm (um) . Minimal fiber type 2 dm (um) . Maximal fiber type 2 dm (um) . Desmin . αB-Crystallin . α-Actinin . Myotilin . Titin . NEM6 41 ± 11 [30–51] (n = 15/15) 7.5 ± 2.6 [4.9–10](n = 15/15) 1.1% ± 0.53 [0.60–1.7](n = 7/15) 2.7% ± 0.70 [2.0–3.4](n = 11/15) id="888">47 ± 15 [32–63](n = 11/13) 27 ± 12 [15–39](n = 11/14) 81 ± 9.0 [72–90] (n = 13) 103 ± 7.6 [96–111] (n = 13) 27 ± 7.0 [20–34] (n = 13) 183 ± 14 [170–197] (n = 13) 84 ± 11 [73–95] (n = 13) 34 ± 11 [24–45] (n = 13) 164 ± 24 [140–188] (n = 13) In rods and in the sarcoplasm of atrophic fibers In the sarcoplasm of some atrophic fibers In rods and in the sarcoplasm of atrophic fibers In rods and in the sarcoplasm of atrophic fibers In proximity of rods and within the sarcoplasm Control 1.0 ± 0.58 [0.44–1.61] (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 48 ± 1.5 [46–49](n = 2) 85 ± 9.4 [76–94] (n = 2) 51 ± 5.4 [46–56] (n = 2) 121 ± 25 [97–146] (n = 2) 92 ± 6.0 [86–98] (n = 2) 57 ± 0.66 [56–58](n = 2) 143 ± 66 [77–209](n = 2) Normal (n = 5) Normal (n = 5) Normal (n = 6) Normal (n = 6) Normal (n = 6) Disease . Routine histological reactions . Immunohistochemistry . . HE/HPhlox . mGT . NADH . ATPase 4.2/fast myosin . Immunoperoxidase . Immunofluorescence . Internalized nuclei (%) . Nuclear clumps (%) . Fiber splitting (%) . Fiber regenera-tion (%) . Rods (%) . Cores (%) . Fiber type 1 (%) . Fiber type 1 dm (um) . Minimal fiber type 1 dm (um) . Maximal fiber type 1 dm (um) . Fiber type 2 dm (um) . Minimal fiber type 2 dm (um) . Maximal fiber type 2 dm (um) . Desmin . αB-Crystallin . α-Actinin . Myotilin . Titin . NEM6 41 ± 11 [30–51] (n = 15/15) 7.5 ± 2.6 [4.9–10](n = 15/15) 1.1% ± 0.53 [0.60–1.7](n = 7/15) 2.7% ± 0.70 [2.0–3.4](n = 11/15) id="888">47 ± 15 [32–63](n = 11/13) 27 ± 12 [15–39](n = 11/14) 81 ± 9.0 [72–90] (n = 13) 103 ± 7.6 [96–111] (n = 13) 27 ± 7.0 [20–34] (n = 13) 183 ± 14 [170–197] (n = 13) 84 ± 11 [73–95] (n = 13) 34 ± 11 [24–45] (n = 13) 164 ± 24 [140–188] (n = 13) In rods and in the sarcoplasm of atrophic fibers In the sarcoplasm of some atrophic fibers In rods and in the sarcoplasm of atrophic fibers In rods and in the sarcoplasm of atrophic fibers In proximity of rods and within the sarcoplasm Control 1.0 ± 0.58 [0.44–1.61] (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 48 ± 1.5 [46–49](n = 2) 85 ± 9.4 [76–94] (n = 2) 51 ± 5.4 [46–56] (n = 2) 121 ± 25 [97–146] (n = 2) 92 ± 6.0 [86–98] (n = 2) 57 ± 0.66 [56–58](n = 2) 143 ± 66 [77–209](n = 2) Normal (n = 5) Normal (n = 5) Normal (n = 6) Normal (n = 6) Normal (n = 6) NEM6 = nemaline myopathy type 6; HE = hematoxylin and eosin; HPhlox = hematoxylin and phloxine; mGT = modified Gömöri trichrome; NADH-TR = reduced nicotinamide adenosine dinucleotide tetrazolium reductase; ATPase = adenosine triphosphatase; dm = diameter; n = number; µm = micrometer; % = percentage; mean ± 2 SD (95% confidence interval). Open in new tab TABLE 3. Histopathologic and Immunohistochemical Features of NEM6 and Controls. Disease . Routine histological reactions . Immunohistochemistry . . HE/HPhlox . mGT . NADH . ATPase 4.2/fast myosin . Immunoperoxidase . Immunofluorescence . Internalized nuclei (%) . Nuclear clumps (%) . Fiber splitting (%) . Fiber regenera-tion (%) . Rods (%) . Cores (%) . Fiber type 1 (%) . Fiber type 1 dm (um) . Minimal fiber type 1 dm (um) . Maximal fiber type 1 dm (um) . Fiber type 2 dm (um) . Minimal fiber type 2 dm (um) . Maximal fiber type 2 dm (um) . Desmin . αB-Crystallin . α-Actinin . Myotilin . Titin . NEM6 41 ± 11 [30–51] (n = 15/15) 7.5 ± 2.6 [4.9–10](n = 15/15) 1.1% ± 0.53 [0.60–1.7](n = 7/15) 2.7% ± 0.70 [2.0–3.4](n = 11/15) id="888">47 ± 15 [32–63](n = 11/13) 27 ± 12 [15–39](n = 11/14) 81 ± 9.0 [72–90] (n = 13) 103 ± 7.6 [96–111] (n = 13) 27 ± 7.0 [20–34] (n = 13) 183 ± 14 [170–197] (n = 13) 84 ± 11 [73–95] (n = 13) 34 ± 11 [24–45] (n = 13) 164 ± 24 [140–188] (n = 13) In rods and in the sarcoplasm of atrophic fibers In the sarcoplasm of some atrophic fibers In rods and in the sarcoplasm of atrophic fibers In rods and in the sarcoplasm of atrophic fibers In proximity of rods and within the sarcoplasm Control 1.0 ± 0.58 [0.44–1.61] (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 48 ± 1.5 [46–49](n = 2) 85 ± 9.4 [76–94] (n = 2) 51 ± 5.4 [46–56] (n = 2) 121 ± 25 [97–146] (n = 2) 92 ± 6.0 [86–98] (n = 2) 57 ± 0.66 [56–58](n = 2) 143 ± 66 [77–209](n = 2) Normal (n = 5) Normal (n = 5) Normal (n = 6) Normal (n = 6) Normal (n = 6) Disease . Routine histological reactions . Immunohistochemistry . . HE/HPhlox . mGT . NADH . ATPase 4.2/fast myosin . Immunoperoxidase . Immunofluorescence . Internalized nuclei (%) . Nuclear clumps (%) . Fiber splitting (%) . Fiber regenera-tion (%) . Rods (%) . Cores (%) . Fiber type 1 (%) . Fiber type 1 dm (um) . Minimal fiber type 1 dm (um) . Maximal fiber type 1 dm (um) . Fiber type 2 dm (um) . Minimal fiber type 2 dm (um) . Maximal fiber type 2 dm (um) . Desmin . αB-Crystallin . α-Actinin . Myotilin . Titin . NEM6 41 ± 11 [30–51] (n = 15/15) 7.5 ± 2.6 [4.9–10](n = 15/15) 1.1% ± 0.53 [0.60–1.7](n = 7/15) 2.7% ± 0.70 [2.0–3.4](n = 11/15) id="888">47 ± 15 [32–63](n = 11/13) 27 ± 12 [15–39](n = 11/14) 81 ± 9.0 [72–90] (n = 13) 103 ± 7.6 [96–111] (n = 13) 27 ± 7.0 [20–34] (n = 13) 183 ± 14 [170–197] (n = 13) 84 ± 11 [73–95] (n = 13) 34 ± 11 [24–45] (n = 13) 164 ± 24 [140–188] (n = 13) In rods and in the sarcoplasm of atrophic fibers In the sarcoplasm of some atrophic fibers In rods and in the sarcoplasm of atrophic fibers In rods and in the sarcoplasm of atrophic fibers In proximity of rods and within the sarcoplasm Control 1.0 ± 0.58 [0.44–1.61] (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 0 (n = 6) 48 ± 1.5 [46–49](n = 2) 85 ± 9.4 [76–94] (n = 2) 51 ± 5.4 [46–56] (n = 2) 121 ± 25 [97–146] (n = 2) 92 ± 6.0 [86–98] (n = 2) 57 ± 0.66 [56–58](n = 2) 143 ± 66 [77–209](n = 2) Normal (n = 5) Normal (n = 5) Normal (n = 6) Normal (n = 6) Normal (n = 6) NEM6 = nemaline myopathy type 6; HE = hematoxylin and eosin; HPhlox = hematoxylin and phloxine; mGT = modified Gömöri trichrome; NADH-TR = reduced nicotinamide adenosine dinucleotide tetrazolium reductase; ATPase = adenosine triphosphatase; dm = diameter; n = number; µm = micrometer; % = percentage; mean ± 2 SD (95% confidence interval). Open in new tab ATPase 4.2 and IHC against fast myosin (n = 13) showed type 1 fiber predominance (81% ± 9.0% [95% CI, 72–90]) in all NEM6 patients (Table 3). The normal fiber checkerboard distribution was lost without evidence of grouping. Type 1 fiber mean diameter was increased (103 ± 7.6 [95% CI, 96–111], p < 0.05) compared to healthy controls. Additionally, type 1 fibers had a highly variable diameter, resulting in a smaller diameter of the most atrophic fibers (27 μm ± 7.0 μm [95% CI, 20–34], p < 0,05) and in an increased diameter of the most hypertrophic fibers (183 μm ± 14 μm [95% CI, 170–197], p < 0,05), compared to healthy controls. Further, the diameter of the most atrophic type 2 fibers was statistically decreased (34 μm ± 11 μm [95% CI, 24–45], p < 0.05) compared to healthy controls. The diameter of the most hypertrophic type 2 fibers was not statistically altered (164 μm ± 24 μm [95% CI, 140–188], p > 0.05) compared to the controls. The mean type 2 fiber diameter was similar to that of healthy controls (84 μm ± 11 [95% CI, 73–95]). The standard deviation of both fiber type 1 and fiber type 2 was significantly increased compared to controls, indicating an increased fiber diameter variation in NEM6 biopsies. Fiber typing was completed using slow and fetal MHCs in 5 samples and confirmed the presence of the above-mentioned findings. Immunohistochemistry Rods were immunoreactive for α-actinin and myotilin (Fig. 2A, B). Strong presence of titin immunoreactivity was constantly observed in sarcoplasmic and subsarcolemmal regions (Fig. 2C–E). In order to explore the degree of sarcomeric integrity around rods clusters and in ring-rods fibers, we performed titin immunohistochemistry/co-immunofluorescence for α-actinin (Fig. 2F–H). Images were analyzed with the confocal microscope to resolve their precise localization. Titin immunoreactivity was found in the areas surrounding the rods and in the sarcoplasm of atrophic fibers, indicating global loss of sarcomeric scaffolding. Scattered desmin-immunoreactive areas were observed in the sarcoplasm and did not have a clear colocalization with rods. Additionally, desmin immunoreactivity was seen in severely atrophic fibers (Fig. 2I). αB-crystallin overexpression (n = 5) was seen in 5.9% ± 3.0% [95% CI, 2.9–8.9] of mainly atrophic fibers (Fig. 2J). Rods were not immunoreactive for αB-crystallin. FIGURE 2. Open in new tabDownload slide Immunohistochemical studies in NEM6 patients. (A) Subsarcolemmal (arrow) and sarcoplasmic (*) α-actinin-immunoreactive areas corresponding to rods. Fibers with subsarcolemmal α-actinin-positive staining represent the ring-rods fibers. Atrophic muscle fibers show intense α-actinin staining (open arrow) (confocal microscopy). (B) Intense myotilin staining in subsarcolemmal (arrow) and sarcoplasmic (*) areas corresponding to rods. Fibers with subsarcolemmal myotilin positive staining represent ring-rods fibers (confocal microscopy). (C) Subsarcolemmal (arrow) and sarcoplasmic (open arrow) titin immunoreactivity (AF555, N2A, N-terminal). (D) Subsarcolemmal (arrow) and sarcoplasmic titin staining, more evident in atrophic fibers (open arrow) (AF 488, M10-1×, C-terminal). (E) Merge of N-terminal and C-terminal titin antibodies staining; subsarcolemmal (arrow) and sarcoplasmic titin staining; the latter is more intense in atrophic fibers (open arrow) (AF555 and AF488, N2A, and M10-×1). (F) Subsarcolemmal (arrow) and sarcoplasmic (open arrow) α-actinin reactivity. (G) Subsarcolemmal (arrow) and sarcoplasmic (open arrow) titin reactivity (AF 488, M10-1×, C-terminal region of titin). (H) Merge of (F) and (G), revealing intense titin reactivity around rods (arrow and open arrow) suggesting secondary sarcomeric disarray. (I) scattered small aggregates immunoreactive for desmin without a clear correspondence to rods (arrow); desmin aggregation in severe atrophic fibers (open arrow) (immunoperoxidase, light microscopy). (J) Absence of αB-crystallin expression in most fibers and slight αB-crystallin overexpression in some atrophic fibers (arrow). FIGURE 2. Open in new tabDownload slide Immunohistochemical studies in NEM6 patients. (A) Subsarcolemmal (arrow) and sarcoplasmic (*) α-actinin-immunoreactive areas corresponding to rods. Fibers with subsarcolemmal α-actinin-positive staining represent the ring-rods fibers. Atrophic muscle fibers show intense α-actinin staining (open arrow) (confocal microscopy). (B) Intense myotilin staining in subsarcolemmal (arrow) and sarcoplasmic (*) areas corresponding to rods. Fibers with subsarcolemmal myotilin positive staining represent ring-rods fibers (confocal microscopy). (C) Subsarcolemmal (arrow) and sarcoplasmic (open arrow) titin immunoreactivity (AF555, N2A, N-terminal). (D) Subsarcolemmal (arrow) and sarcoplasmic titin staining, more evident in atrophic fibers (open arrow) (AF 488, M10-1×, C-terminal). (E) Merge of N-terminal and C-terminal titin antibodies staining; subsarcolemmal (arrow) and sarcoplasmic titin staining; the latter is more intense in atrophic fibers (open arrow) (AF555 and AF488, N2A, and M10-×1). (F) Subsarcolemmal (arrow) and sarcoplasmic (open arrow) α-actinin reactivity. (G) Subsarcolemmal (arrow) and sarcoplasmic (open arrow) titin reactivity (AF 488, M10-1×, C-terminal region of titin). (H) Merge of (F) and (G), revealing intense titin reactivity around rods (arrow and open arrow) suggesting secondary sarcomeric disarray. (I) scattered small aggregates immunoreactive for desmin without a clear correspondence to rods (arrow); desmin aggregation in severe atrophic fibers (open arrow) (immunoperoxidase, light microscopy). (J) Absence of αB-crystallin expression in most fibers and slight αB-crystallin overexpression in some atrophic fibers (arrow). Electron Microscopy Ultrastructural alterations consisted of rods (n = 16, 89%) and cores (n = 15, 83%) (Table 4; Fig. 3), confirming the rod-core scenario observed in light microscopy. Rods were gathered in large clusters or dispersed in the sarcoplasm. Their shape was either elongated, ovoid, squared, or round. In general, they had a longitudinal orientation. Rods clusters spanned multiple sarcomeres and contained nemaline bodies with different shapes and orientations. The clusters provoked a focal disruption of sarcomeres. Isolated rods showed to arise directly from the Z-line, and had an elongated shape and were randomly dispersed in the cytoplasm (Fig. 3A). Large clusters presented abundant filamentous protein material surrounding the rods (Fig. 3B). They spanned 1 or 2 sarcomeres. Star-shaped rods clusters seen with mGT were recognizable to some extent by electron microscopy (data not shown). All rods were composed of compacted Z-line material and, at very high magnification, they showed the typical lattice structures composed by a dense net of orthogonally disposed filaments (Fig. 3C). Areas of completely disorganized sarcomere structure with loosely packed thin filaments and small fragments of dotty Z-line different from full-blown rods were also present. We did not observe intranuclear rods. The Z-lines in the proximity of small rods were undulated and thickened. FIGURE 3. Open in new tabDownload slide Electron micrographs showing subsarcolemmal and sarcoplasmic rods, cores and granulofilamentous material, longitudinal and transverse sections (A). Clusters of sarcoplasmic rods (arrow); the overall sarcomeric structure is well-preserved (open arrow). (B) Subsarcolemmal rods cluster (arrow). (C) High magnification of a rod showing the typical lattice structure composed by a dense net of orthogonally disposed filaments. (D) Accumulation of Z-line material, loss of the sarcomeric structure and absence of mitochondria corresponding to a core lesion (indicated by the arrows). (E) Granulofilamentous protein material (arrow) that is spatially not related to rods. (F) Dense fly-like osmiophilic granulofilamentous material corresponding to desmin. FIGURE 3. Open in new tabDownload slide Electron micrographs showing subsarcolemmal and sarcoplasmic rods, cores and granulofilamentous material, longitudinal and transverse sections (A). Clusters of sarcoplasmic rods (arrow); the overall sarcomeric structure is well-preserved (open arrow). (B) Subsarcolemmal rods cluster (arrow). (C) High magnification of a rod showing the typical lattice structure composed by a dense net of orthogonally disposed filaments. (D) Accumulation of Z-line material, loss of the sarcomeric structure and absence of mitochondria corresponding to a core lesion (indicated by the arrows). (E) Granulofilamentous protein material (arrow) that is spatially not related to rods. (F) Dense fly-like osmiophilic granulofilamentous material corresponding to desmin. TABLE 4. Ultrastructural features of NEM6 Patients. Ultrastructural feature . Number of affected NEM6 patients . Rods (subsarcolemmal and sarcoplasmic; groups and individually scattered) 16/18 (88.9%) Cores 15/18 (83.3%) Granulofilamentous protein material 9/18 (50%) Ultrastructural feature . Number of affected NEM6 patients . Rods (subsarcolemmal and sarcoplasmic; groups and individually scattered) 16/18 (88.9%) Cores 15/18 (83.3%) Granulofilamentous protein material 9/18 (50%) Open in new tab TABLE 4. Ultrastructural features of NEM6 Patients. Ultrastructural feature . Number of affected NEM6 patients . Rods (subsarcolemmal and sarcoplasmic; groups and individually scattered) 16/18 (88.9%) Cores 15/18 (83.3%) Granulofilamentous protein material 9/18 (50%) Ultrastructural feature . Number of affected NEM6 patients . Rods (subsarcolemmal and sarcoplasmic; groups and individually scattered) 16/18 (88.9%) Cores 15/18 (83.3%) Granulofilamentous protein material 9/18 (50%) Open in new tab The peculiar ring-rods disposition was recognizable in some fibers (Fig. 4). Additionally, normally appearing mitochondria were found intermingled with subsarcolemmal rods (Fig. 4), reflecting the presence of an intense subsarcolemmal NADH-TR and SDH rim observed in light microscopy. FIGURE 4. Open in new tabDownload slide Electron micrographs of a ring-rods fiber. At the center presence of cross section of an entire muscle fiber at low magnification showing the presence of dotty osmiophilic material corresponding to rods in proximity of the light blue circle. The presence of rods with their subsarcolemmal disposition is confirmed in the 4 inset images at higher magnification corresponding to the areas indicated by the arrows. Note the presence of normally structured mitochondria intermingled with the rods. FIGURE 4. Open in new tabDownload slide Electron micrographs of a ring-rods fiber. At the center presence of cross section of an entire muscle fiber at low magnification showing the presence of dotty osmiophilic material corresponding to rods in proximity of the light blue circle. The presence of rods with their subsarcolemmal disposition is confirmed in the 4 inset images at higher magnification corresponding to the areas indicated by the arrows. Note the presence of normally structured mitochondria intermingled with the rods. Cores (15 biopsies; 83%) were randomly located in the sarcoplasm, had a variable diameter, spanned several sarcomeres, but never the entire fiber length. The cores contained compacted myofibrils, accumulated Z-line material, and the mitochondria were set apart from the core lesions (Fig. 3D). Nine biopsies (50%) showed areas containing granulofilamentous protein material that appeared less osmiophilic than Z-line material (Fig. 3E, F). This granulofilamentous protein surcharge was spatially distinct from the areas containing rods (Fig. 3E). However, rods cluster, myofibrillar disorganization, and granulofilamentous protein material were occasionally observed next to each other (data not shown). In a few fibers the sarcomeric structure was completely replaced by granulofilamentous protein material. In Patient 13, the only patient harboring the c.1170G>C, p.(Lys390Asn) variant, we identified highly dense osmiophilic material intermingled with less dense granulofilamentous protein material. The latter strongly resembled desmin (Fig. 3F). The presence of internalized nuclei and nuclear clumps was confirmed by electron microscopy in all biopsies. In addition, we found lipofuscin in 12 biopsies, some degree of glycogen accumulation in 6 biopsies, and mitochondria proliferation in 3. We observed swollen mitochondria in 2 biopsies. Eventually, we did not observe any enlargement of the sarcotubular system. Morphological and Clinical Correlation No significant morphological/clinical correlation was found between morphologic lesions (internalized nuclei, nuclear clumps, splitting, regeneration, rods, cores, type 1 fiber predominance, and the percentage of desmin or αB-crystallin-immunoreactive fibers) and global or quadriceps muscle weakness at the moment of biopsy and at the most recent clinical examination. DISCUSSION This is the first systematic study that thoroughly investigates the myopathological features of the ultrarare KBTBD13 congenital myopathy in 18 Dutch patients. KBTBD13 congenital myopathy has been classified among the NEMs group due to the presence of rods in the muscle biopsies of affected patients. Our study confirms rods as the main morphological finding, being present in 16 biopsies (89%). In addition, we identified the coexistence of rods and cores, and nuclear clumps as specific findings, and demonstrated ring-rods fibers and granulofilamentous protein aggregation in a subset of biopsies. Ultrastructurally, rods harbored a typical lattice structure composed by an orthogonal and perpendicular disposition of filaments as a dense net (Fig. 3C), which is not dissimilar from rods found in NEM2 (18) and NEM3 (ACTA1-related nemaline myopathy) (personal observation). By light microscopy, rods were disposed in a star-shaped manner in 6 patients (Fig. 1C), and they sometimes had a subsarcolemmal rim disposition, leading to the appearance of ring-rods fibers (Fig. 4). By electron microscopy we demonstrated that these regions harbor normally shaped mitochondria intermingled with rods (Fig. 4, insets). The presence of ring-rods fibers demarcates KBTBD13-related myopathy from other NEMs (4, 18, 20, 21). These ring-rods fibers differed from the sarcoplasmic circular regions (clusters of rods) previously described by Gommans et al (12). Although not constantly found, ring-rods fibers might be considered a KBTD13 histologic marker, such as the rods with multiple brick-like or stacks of Z-line found in LMOD3-related NEM (3). From an immunohistochemical standpoint, rods were typically immunoreactive for α-actinin and myotilin (Fig. 2). As mutated KBTBD13 does not interact properly with thin filaments/actin, we can speculate that aggregation of Z-disk proteins myotilin and α-actinin in rods might be indirectly caused by the binding of mutant KBTBD13 to the thin-filament protein actin (15). The demonstration of KBTBD13 inside rods was not performed as KBTBD13 protein could not be visualized using commercially available KBTBD13 primary antibodies in our previous studies (15). Co-immunofluorescence studies including antibodies directed against the huge sarcomeric protein titin showed relative spared sarcomeric scaffolding in proximity of rods (Fig. 2) and demonstrated titin-immunoreactive material around them. Cores, present in 83% of biopsies, were the second most important histopathologic lesion. They were generally single, round-shaped, and variably sized. They showed poorly or, less frequently sharply, demarcated borders, and were found in both type 1 and type 2 fibers. Ultrastructurally, they contained compacted myofibrils, accumulation of Z-line material and lacked mitochondria (Fig. 3D). They occupied large parts of the muscle fibers, were both structured and unstructured, and resembled to some extent to typical cores found in RYR1-related CM (22). However, they never spanned the entire muscle fiber length as the typical central (23). The association of nemaline bodies/rods with well-defined cores within separate muscle fiber regions has been described in familial and isolated cases presenting a core-rod myopathy (24–31). Due to the outstanding coexistence of rods and cores in our cohort, we argue to classify KBTBD13-related congenital myopathy as a rods-core myopathy. Nuclear clumps are the end product of severe muscle fiber atrophy and may be a sign of denervation without reinnervation (32). We observed nuclear clumps in all biopsies (Fig. 1A). Our patients had normal reflexes, no sensory disturbances, no distal atrophy, and no signs of denervation on electromyography, arguing against neuropathic involvement (33). Due to the relatively advanced age of our patients at biopsy, one could argue that nuclear clumps reflect an age-related unspecific finding. In our experience, we did not disclose the presence of elevated incidence of nuclear clumps associated with age. More importantly, there was no correlation between the number of nuclear clumps and the patient’s age at biopsy in our cohort (spearman’s correlation, p > 0.05). We therefore suggest that the presence of nuclear clumps with rods cores might direct the myopathologist towards a KBTBD13 diagnosis. Desmin is an intermediate filament protein that integrates the sarcolemma, Z-disk, and nuclear membrane in sarcomeres (34). Aggregates immunoreactive for desmin can be found in multiple conditions in which the sarcomere structure is disrupted, including desmin-related myofibrillar myopathies (35). Previous NEM6 histopathologic studies have shown different results on desmin localization and (over)expression (16, 17). By immunohistochemistry we showed desmin overexpression in the sarcoplasm of severe atrophic fibers possibly related to regeneration (36). Of note, desmin Western blot analysis in a muscle biopsy from a Spanish NEM6 patient also showed increased levels of desmin (personal communication from Dr. Montse Olivé). Desmin material might be indirectly caused by mutated KBTBD13. αB-crystallin is a protein that is associated with actin microfilaments at Z-disks and is a member of the heat shock proteins. Enhanced sarcoplasmic αB-crystallin expression is seen in myofibrillar myopathies (38); it was described in muscle biopsy from 2 siblings showing increased αB-crystallin expression in hyaline masses accompanying nemaline rods (39), and in a mouse model of actin NEM. We also identified αB-crystallin material in some but not all atrophic fibers, being possibly a response to disruption of the myofibrillar architecture, consistent with the chaperone role of αB-crystallin for desmin (40, 41). Although we did not find immunohistochemical criteria for KBTBD13-related myopathy among the myofibrillar myopathies, ultrastructural analysis revealed the presence of protein aggregates composed of granulofilamentous protein material in 9 patients. This granulofilamentous material, found in areas separated from rods clusters, suggested a primary granulomatous protein deposition distinct from the secondary cytoskeletal disarray surrounding rods and cores (Fig. 3E). Protein material was constituted by granular Z-line-like material, i.e. it was less electrodense than that found in desminopathies and αB-crystallinopathies (42). We also showed a preponderating filamentous component that is in contrast to the wider granular component in desminopathies (35). Furthermore, in contrast to desminopathies, we did not observe band-like distribution of electrodense granulofilamentous protein material in the subsarcolemmal space or surrounding the myonucleus (42). Only P13 biopsy had protein aggregates strongly resembling desmin deposition (Fig. 3F). This patient is also unique from a genetic standpoint harboring the c.1170G>C, p.(Lys390Asn) variant. We conclude that the protein aggregation differs from the other patients’ muscles harboring different mutations. Since 15 NEM6 patients harbored the same Dutch founder variant in the KBTBD13 gene, our study presents a unique, detailed muscle pathology phenotyping of patients affected by this founder culprit. However, the translation of our findings on muscle pathology to patients carrying different mutations in the KBTBD13 gene is uncertain and needs further analysis with additional patients. NEM6 patients showed muscle slowness and slowly progressive neck flexor and proximal muscle weakness starting in childhood. We failed to identify a significant correlation between NEM6 histopathological features and muscle weakness severity (43, 44). Lack of data on muscle strength, a large discrepancy between the age of some patients at the moment of biopsy and at clinical examination, and the retrospective, non-standardized character of data collection might cause a possible clinic-morphologic correlation to remain undissolved. Clinically, the neck flexor muscles, and not the quadriceps muscles, were most severely affected, suggesting neck flexor muscle as a more representative muscle for assessing clinical-morphological correlation. In conclusion, we extensively described the morphological features of a cohort of 18 KBTBD13 patients by histochemistry, immunohistochemistry, and electron microscopy. The presence of ring-rods fibers, nuclear clumps and granulofilamentous protein material are proposed as novel histopathologic markers. Due to the coexistence of rods and cores in most NEM6 patients, we suggest classifying KBTBD13-related congenital myopathy as a rod-core myopathy. ACKNOWLEDGMENTS The study was conducted at the University of Versailles-Saint-Quentin-en-Yvelines (France) in close collaboration with Radboud University Medical Center (The Netherlands). Special thanks go to Hôpital Henri Mondor (France) for enabling routine histopathological reactions. We are grateful to Dr Montse Olivé (IDIBELL-Hospital de Bellvitge, Hospitalet de Llobregat, Barcelona, Spain) for sharing her expertise on muscle pathology and for her critical comments on the manuscript. We thank Isabelle Richard (Généthon, Évry, France) for antibodies against M10.1 and N2A. The present work has benefited from Imagerie-Gif core facility supported by l’Agence Nationale de la Reserche (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBioImaging; ANR-11-IDEX-0003-02/Saclay Plant Sciences). This work was financially supported by the Radboud Honours Academy of the Radboud University Nijmegen (The Netherlands). JW is funded by a NOW-Rubicon grant (452173126). 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TI - NEM6, KBTBD13-Related Congenital Myopathy: Myopathological Analysis in 18 Dutch Patients Reveals Ring Rods Fibers, Cores, Nuclear Clumps, and Granulo-Filamentous Protein Material
JF - Journal of Neuropathology & Experimental Neurology
DO - 10.1093/jnen/nlab012
DA - 2021-03-22
UR - https://www.deepdyve.com/lp/oxford-university-press/nem6-kbtbd13-related-congenital-myopathy-myopathological-analysis-in-jFF4x4UBBm
SP - 366
EP - 376
VL - 80
IS - 4
DP - DeepDyve
ER -