Congenital Nemaline Myopathy with Dense Protein Masses

To the Editor: Nemaline myopathies (NM) are a heterogeneous group of rare congenital muscle disorders characterized by hypotonia, muscle and facial weakness, delayed motor milestones, and skeletal deformities with nemaline bodies (rods) in the muscle biopsy (1). To date, 14 genes have been linked to NM: ACTA1, NEB, TPM2, TPM3, TNNT1, KBTBD13, CFL2, KLHL40, KLHL41, LMOD3, MYO18B, MYPN, RYR3, and TNNT3 (2–5). These genes encode skeletal muscle thin filament proteins, their interactors with, or their regulators, such as Kelch domain-associated proteins and an unconventional myosin. Nebulin gene (NEB) is the most commonly mutated and is thought to account for approximately 50% of genetically diagnosed cases of NM (4). However, genotype-morphological phenotype correlations have been difficult to establish in NEB-related myopathy (6). In previous work, we have shown that clinical severity correlates with the degree of sarcomeric dissociation and contractility efficiency but rod burden does not seem to show a positive correlation with clinical severity (7). In this report, we describe a particular histologic lesion observed in the muscle biopsies of 5 unrelated patients, 3 with NEB-NM, 1 with TPM2-NM, and 1 without genetic characterization. The lesion consists of round-shaped dense protein masses (DPM) found in the center of the sarcoplasm separated from clustered rods and extending longitudinally several microns (Fig. 1). On ATPase staining, type 1 fiber predominance or uniformity was observed and the areas corresponding to DPM were devoid of reaction (not shown). By electron microscopy, the DPM are composed of Z-band sarcomeric protein mainly displaced to the subsarcolemmal space (Fig. 1M–O). Clinically, the patients showed variable onset from early childhood, with muscle weakness, skeletal deformities, and delayed motor milestones, to late-onset-myopathy beginning in the third decade as progressive generalized amyotrophy and muscle weakness (Table). All patients presented with a moderate restrictive respiratory impairment without cardiac involvement. Histological and ultrastructural findings in P1, P2, P4 deltoid muscle biopsies. Representative cross-sections of the deltoid biopsy of P1 (A–D), P2 (E–H), and P4 (I–L). With hematoxylin and eosin (HE) (A, E, I) and modified Gomori’s trichrome (GT) staining (B, F, J), variable numbers of fibers showed a round, central, or eccentric dense aggregate (dense protein masses [DPM]) (black arrows), clearly distinguishable from the rods (yellow arrows) and nuclei (green arrows). Note in J, the DPM in continuity with clusters of intracytoplasmic rods. With NADH-TR (NADH) (C, G, K) areas of intermyofibrillary network disorganization and absence of oxidative staining correspond to protein aggregates. DPM showed positive immunostaining to αB-crystallin (αBC) (D, H, L) (black arrows), myotilin and desmin (not shown). Panels A, B, E, F, and I–K are serial sections. Scale bar in L = 20 µm. Electron microscopic features of P1 muscle (N–O). Transverse section through a dense cylindrical mass observed as a well-defined, round formation, centrally placed on the fiber (M). Longitudinal section showing a DPM extending along several sarcomeres, paralleling the longitudinal axis of the fiber (N). DPM consisted of aggregates of thin filaments, Z-line proteins, and granulofilamentous desmin aggregates, intermingled with rods. A close-up image of the box depicted in (N) shows regions were DPM intercalated with rods and internalized nuclei in the center of the fiber. Autophagic vacuoles are also seen in the periphery (O). Clinical, Pathologic and Genetic Features of the Patients Childhood. Facial and bulbar weakness. Generalized weakness distal≫proximal. Pes Cavus. CPK: 29 UI/L. EMG: myopathic. Cardio: N, CV: 68%, restrictive. NEB Intron 151* c.22266 + 1_22266 + 9delGTAAGTGAT p.Asn5650_Asp5686del Exon 55* c.7431 + 1916_7536 + 372del p.Arg2478_Asp2512del Compound Heterozygous NEB Intron 17† c.1569 + 1G>A p.His491_Asp523del Exon 176† c.24606del p.Ala8203Glnfs*13 Compound Heterozygous TPM2 c.20_22delAGA p.Lys7del Heterozygous Childhood. Facial and bulbar weakness. Generalized weakness distal≫proximal. Pes Cavus. CPK: 29 UI/L. EMG: myopathic. Cardio: N, CV: 68%, restrictive. NEB Intron 151* c.22266 + 1_22266 + 9delGTAAGTGAT p.Asn5650_Asp5686del Exon 55* c.7431 + 1916_7536 + 372del p.Arg2478_Asp2512del Compound Heterozygous NEB Intron 17† c.1569 + 1G>A p.His491_Asp523del Exon 176† c.24606del p.Ala8203Glnfs*13 Compound Heterozygous TPM2 c.20_22delAGA p.Lys7del Heterozygous Histol, Histological findings; NM, nemaline myopathy features; T1U, type 1 fiber uniformity; T1D, type 1 fiber predominance; DPM, dense protein masses; NI, internalized nuclei; CR, Congo Red; IHQ, immunohistochemistry; Cardio, cardiologic assessment; WPW, Wolff-Parkinson-White syndrome. Family 52 in Lehtokari et al (8). Patient P14 in Malfatti et al (7). Clinical, Pathologic and Genetic Features of the Patients Childhood. Facial and bulbar weakness. Generalized weakness distal≫proximal. Pes Cavus. CPK: 29 UI/L. EMG: myopathic. Cardio: N, CV: 68%, restrictive. NEB Intron 151* c.22266 + 1_22266 + 9delGTAAGTGAT p.Asn5650_Asp5686del Exon 55* c.7431 + 1916_7536 + 372del p.Arg2478_Asp2512del Compound Heterozygous NEB Intron 17† c.1569 + 1G>A p.His491_Asp523del Exon 176† c.24606del p.Ala8203Glnfs*13 Compound Heterozygous TPM2 c.20_22delAGA p.Lys7del Heterozygous Childhood. Facial and bulbar weakness. Generalized weakness distal≫proximal. Pes Cavus. CPK: 29 UI/L. EMG: myopathic. Cardio: N, CV: 68%, restrictive. NEB Intron 151* c.22266 + 1_22266 + 9delGTAAGTGAT p.Asn5650_Asp5686del Exon 55* c.7431 + 1916_7536 + 372del p.Arg2478_Asp2512del Compound Heterozygous NEB Intron 17† c.1569 + 1G>A p.His491_Asp523del Exon 176† c.24606del p.Ala8203Glnfs*13 Compound Heterozygous TPM2 c.20_22delAGA p.Lys7del Heterozygous Histol, Histological findings; NM, nemaline myopathy features; T1U, type 1 fiber uniformity; T1D, type 1 fiber predominance; DPM, dense protein masses; NI, internalized nuclei; CR, Congo Red; IHQ, immunohistochemistry; Cardio, cardiologic assessment; WPW, Wolff-Parkinson-White syndrome. Family 52 in Lehtokari et al (8). Patient P14 in Malfatti et al (7). Patients P1, P2, and P3 were previously reported in (1) and (7), and harbored, respectively, the compound heterozygote pathogenic variants p. N5650_D5686del in intron 151 and p. R2478_D2512del on exon 55 (P1 and P2), and p. H491_D523del in intron 17 and p. A8203Glnfs*13 in exon 176 in NEB (Table). P5 has the c.20_22del, p. Lys7del in tropomyosin 2 gene (TPM2) at a heterozygous level (9, 10). In contrast, P4 is genetically unsolved, despite exhaustive analysis of known genes in NM. Similar histopathological findings were reported by Selcen et al (11) in two patients with a different clinical picture reported as “familial cardioneuromyopathy with hyaline masses and nemaline rods” without genetic diagnosis. The described protein aggregates were congophilic and labeled for desmin and αB-crystallin. In the patients described here, the dense protein cylindric masses seemingly contained desmin, αB-crystallin and myotilin. Congo red staining was only performed in P4 and the aggregates were Congo red-negative but protein labeling of the DPM was not performed. We believe that the DPM are the same finding as that described by Selcen et al (11), but that the term “dense protein masses,” rather “hyaline,” describes more accurately the characteristics of the DPM, which are not translucent. The mechanism leading to the association of the dense protein accumulations and rods in our NEB and TPM2 NM remains elusive. We hypothesize that aggregation of diverse proteins could be the consequence of impaired extralysosomal protein degradation. Despite the mechanism underlying the protein aggregation, the DPM impair contractility thus contributing to cytoskeletal disorganization and nuclear morphology (12). In conclusion, we describe here the association of DPM and nemaline rods linked to NEB and TPM2. This report enlarges the histopathological spectrum NM and adds another histological phenotype subgroup. Publication of this article was partially funded by Association Institut de Myologie (AIM) and Association française contre les myopathies-Téléthon (AFM-Téléthon) financial support to the Neuromuscular Morphologic Unit to NBR and grants FONDECYT 1151383 and Travel Grant from the Dirección de Docencia y Desarrollo, Clínica Dávila to JAB. The authors have no duality or conflicts of interest to declare.