# Congenital Nemaline Myopathy with Dense Protein Masses

Jorge Bevilacqua, Edoardo Malfatti, Clémence Labasse, Guy Brochier, Angeline Madelaine, Emmanuelle Lacène, Bérénice Doray, Pascal Laforêt, Bruno Eymard, John Rendu, et al.

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## To cite this version:

Jorge Bevilacqua, Edoardo Malfatti, Clémence Labasse, Guy Brochier, Angeline Madelaine, et al. Congenital Nemaline Myopathy with Dense Protein Masses. Journal of Neuropathology and Experimental Neurology, 2022, 81 (4), pp.304-307. 10.1093/jnen/nlab139. inserm-04877755

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Distributed under a Creative Commons Attribution 4.0 International License
Congenital Nemaline Myopathy with Dense Protein Masses

Jorge A. Bevilacqua ID, MD, PhD<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Edoardo Malfatti, MD, PhD<fnref n="4" />, Clémence Labasse, BSc<fnref n="5" />,
Guy Brochier, PhD<fnref n="5" />,<fnref n="6" />, Angeline Madelaine, BSc<fnref n="5" />,<fnref n="6" />, Emmanuelle Lacène, BSc<fnref n="5" />,<fnref n="6" />, Bérénice Doray, MD<fnref n="7" />,
Pascal Laforêt, MD, PhD<fnref n="8" />,<fnref n="9" />, Bruno Eymard, MD, PhD<fnref n="6" />, John Rendu ID, PhD<fnref n="10" />, and
Norma B. Romero ID, MD, PhD<fnref n="5" />,<fnref n="6" />,<fnref n="11" />

Correspondence to: Norma Beatriz Romero, MD, PhD, Myology Institute, Sorbonne Université, GHU La Pitie-Salpêtrière, 75013 Paris, France; E-mail: nb.romero@institut-myologie.org

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.

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

<footnote n="1">¹Laboratorio de Patología Muscular, Departamento de Neurología y Neurocirugía, Clínica D'avila, Santiago, Chile</footnote>

<footnote n="2">²Departamento Neurología y Neurocirugía, Unidad Neuromuscular, Hospital Clínico Universidad de Chile, Santiago, Chile</footnote>

<footnote n="3">³Departamento de Anatomía y Medicina Legal, Facultad de Medicina, Universidad de Chile, Santiago, Chile</footnote>

<footnote n="4">⁴APHP, Neuromuscular Reference Center Nord-Est-Ile-de-France, Henri Mondor Hospital, Université Paris Est, U955, INSERM, Cr'eteil, IMRB, France</footnote>

<footnote n="5">⁵Institut de Myologie, Neuromuscular Morphology Unit, Groupe Hospitalier Pitie-Salpêtrière, Paris, France</footnote>

<footnote n="6">⁶AP-HP, Centre de R'ef'erence de Pathologie Neuromusculaire Nord/Est/Ile de France, Groupe Hospitalier Pitie-Salpêtrière, Paris, France</footnote>

<footnote n="7">⁷CHU de La Reunion, Hôpital Felix Guyon Pole Biologie Nord, Service G'en'etique Saint Denis, France</footnote>

<footnote n="8">⁸Service Neurologie Médicale, Centre de R'ef'erence Maladies Neuromusculaire Paris-Est-Ile de France, CHU Raymond-Poincaré Paris Ouest</footnote>

<footnote n="9">⁹INSERM Handicap Neuromusculaire, UFR des sciences de la santé Simone Veil, Université Versailles-Saint-Quentin-en-Yvelines</footnote>

<footnote n="10">¹⁰Univ. Grenoble Alpes, Inserm, U1216, CHU Grenoble Alpes, Grenoble Institut Neurosciences, Grenoble, 38000, France</footnote>

<footnote n="11">¹¹Sorbonne Université, INSERM, Institut de Myologie, Centre de recherche en Myologie, GHU Pitie-Salpêtrière, Paris, France</footnote>
<table><caption>TABLE. Clinical, Pathologic and Genetic Features of the Patients</caption><thead><tr><th colspan="3">Epidemiology</th><th colspan="3">Biopsy Features</th><th colspan="2">Clinical Features and Genetics</th></tr><tr><th>Patient</th><th>Sex/ Current age</th><th>Origin</th><th>Muscle/Biopsy Age</th><th>Histology</th><th>IHQ</th><th>Electron Microscopy</th><th>Age at onset / Clinical Phenotype</th><th>Mutation:</th></tr></thead><tbody><tr><td>P1</td><td>M/27 year</td><td>France</td><td>Deltoid 26 year</td><td>NM, T1U, DPM,</td><td>Desmin + αB-crystallin + Myotilin +</td><td>Subsarcolemmal rods, central DPM, desmin aggregates</td><td>Childhood. Facial and bulbar weakness. Generalized weakness distal proximal. Pes Cavus. CPK: 29 UI/L. EMG: myopathic. Cardio: N, CV: 68%, restrictive.</td><td>NEB<br>Intron 151*<br>c.22266 + 1_22266 + 9delGTAAGTGAT<br>p.Asn5650_Asp5686del<br>Exon 55*<br>c.7431 + 1916_7536 + 372del<br>p.Arg2478_Asp2512del</td></tr><tr><td>P2</td><td>M/29 year</td><td>France</td><td>Deltoid 29 year</td><td>NM, NI, T1D, DPM,</td><td>Desmin + αB-crystallin + Myotilin +</td><td>Subsarcolemmal rods, central DPM, desmin aggregates</td><td>Asymptomatic. Mild distal lower limb weakness, playing sports. EMG: mild chronic myogenic signs. Cardio: N.</td><td>Compound Heterozygous</td></tr><tr><td>P3</td><td>F/41 year</td><td>France</td><td>Deltoid 21 yr</td><td>NM, T1D, DPM,</td><td>Not done</td><td>Subsarcolemmal rods, central DPM, desmin aggregates</td><td>Mild congenital NM. Frequent falls. Difficulties in running, rising stairs. Jaw contractures. Nasal voice. Elongated face. Axial weakness with difficulties in neck flexion. Asymmetrical distal weakness with foot drop (right>left). CPK: 24 UI/L. Cardio: N. CV: 40% NIV</td><td>NEB<br>Intron 17†<br>c.1569 + 1G>A<br>p.His491_Asp523del<br>Exon 176†<br>c.24606del<br>p.Ala8203Glnfs*13</td></tr><tr><td>P4</td><td>M/58 year</td><td>Reunion Island</td><td>Deltoid 56 year</td><td>NM, T1U, DPM, CR (—)</td><td>Desmin + αB-crystallin + Myotilin +</td><td>Subsarcolemmal rods, central DPM, desmin aggregates</td><td>Childhood. Kyphoscoliosis. Deafness. Pes Cavus. Generalized amyotrophy, weakness distal>proximal. CPK: N. EMG: myopathic. Cardio: N. FEV1: 73%, restrictive. NIV</td><td>Compound Heterozygous<br>None found</td></tr><tr><td>P5</td><td>M/67 year</td><td>Portugal</td><td>Deltoid 37 year</td><td>NM, NI, T1D, DPM, ring fibers</td><td>Note done</td><td>Rods, central DPM, desmin aggregates, annular fibers</td><td>At 35 year, fatigability and lower limb pain after efforts. Cramps. Distal leg amyotrophy. CPK: 3xN. Cardio: Asymptomatic WPW.</td><td>TPM2<br>c.20_22delAGA<br>p.Lys7del<br>Heterozygous</td></tr></tbody></table>

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.

<footnote>*Family 52 in Lehtokari et al (8).</footnote>

<footnote>†Patient P14 in Malfatti et al (7).</footnote>
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FIGURE 1. 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 aB-crystallin (aBC) (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 ¼ 20 mm. 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 where DPM intercalated with rods and internalized nuclei in the center of the fiber. Autophagic vacuoles are also seen in the periphery (O).

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 aB-crystallin. In the patients described here, the dense protein cylindric masses seemingly contained desmin, aB-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.
## FUNDING

Publication of this article was partially funded by Association Institut de Myologie (AIM) and Association française contre les myopathies-T'el'ethon (AFM-T'el'ethon) financial support to the Neuromuscular Morphologic Unit to NBR and grants FONDECYT 1151383 and Travel Grant from the Dirección de Docencia y Desarrollo, Clínica D'avila to JAB.

## COMPETING INTERESTS

The authors have no duality or conflicts of interest to declare.

## REFERENCES

1. Romero NB, Sandaradura SA, Clarke NF. Recent advances in nemaline myopathy. Curr Opin Neurol 2013;26:519-26

2. Radke J, Stenzel W, Goebel HH. Recently Identified congenital myopathies. Semin Pediatr Neurol 2019;29:83-90

3. Sewry CA, Laitila JM, Wallgren-Pettersson C. Nemaline myopathies: A current view. J Muscle Res Cell Motil 2019;40:111-26

4. Malfatti E, Romero NB. Nemaline myopathies: State of the art. Rev Neurol (Paris) 2016;172:614-9

5. Sandaradura SA, Bournazos A, Mallawaarachchi A, et al. Nemaline myopathy and distal arthrogryposis associated with an autosomal recessive TNNT3 splice variant. Hum Mutat 2018;39: 383-8

6. Maggi L, Scoto M, Cirak S, et al. Congenital myopathies—Clinical features and frequency of individual subtypes diagnosed over a 5-year period in the United Kingdom. Neuromuscul Disord 2013;23: 195-205

7. Malfatti E, Lehtokari VL, Bo¨ hm J, et al. Muscle histopathology in nebulin-related nemaline myopathy: Ultrastructural findings correlated to disease severity and genotype. Acta Neuropathol Commun 2014;2:44

8. Lehtokari V-L, Pelin K, Sandbacka M, et al. Identification of 45 novel mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. Hum Mutat 2006;27:946-56

9. Mokbel N, Ilkovski B, Kreissl M, et al. K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity. Brain 2013;136:494-507

10. Davidson AE, Siddiqui FM, Lopez MA, et al. Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies. Brain 2013;136:508-21

11. Selcen D, Krueger BR, Engel AG. Familial cardioneuromyopathy with hyaline masses and nemaline rods: A novel phenotype. Ann Neurol 2002;51:224-34

12. Ross JA, Levy Y, Ripolone M, et al. Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy. Acta Neuropathol 2019;138:477-95