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Tóm tắt nội dung (trích từ tài liệu gốc): J Neuropathol Exp Neurol Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021 Vol. 77, No. 12, December 2018, pp. 1101�1114 doi: 10.1093/jnen/nly095 ORIGINAL ARTICLE Loss of Sarcomeric Scaffolding as a Common Baseline Histopathologic Lesion in Titin-Related Myopathies Rainiero A vila-Polo, MD, Edoardo Malfatti, MD, PhD, Xavie`re Lornage, MSc, Chrystel Cheraud, MD, Isabelle Nelson, PhD, Juliette Nectoux, PharmD, PhD, Johann Bo�hm, PhD, Raphael Schneider, MSc, Carola Hedberg-Oldfors, PhD, Bruno Eymard, MD, PhD, Soledad Monges, MD, Fabiana Lubienieck
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J Neuropathol Exp Neurol Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
Vol. 77, No. 12, December 2018, pp. 1101�1114
doi: 10.1093/jnen/nly095
ORIGINAL ARTICLE
Loss of Sarcomeric Scaffolding as a Common Baseline
Histopathologic Lesion in Titin-Related Myopathies
Rainiero A vila-Polo, MD, Edoardo Malfatti, MD, PhD, Xavie`re Lornage, MSc, Chrystel Cheraud, MD,
Isabelle Nelson, PhD, Juliette Nectoux, PharmD, PhD, Johann Bo�hm, PhD, Raphael Schneider, MSc,
Carola Hedberg-Oldfors, PhD, Bruno Eymard, MD, PhD, Soledad Monges, MD,
Fabiana Lubieniecki, MD, Guy Brochier, PhD, Mai Thao Bui, BSc, Angeline Madelaine, BSc,
Clemence Labasse, BSc, Maud Beuvin, MSc, Emmanuelle Lace`ne, MSc, Anne Boland, PhD,
Jean-Franc�ois Deleuze, PhD, Julie Thompson, PhD, Isabelle Richard, PhD,
Ana Lia Taratuto, MD, PhD, Bjarne Udd, MD, PhD, France Leturcq, PharmD, Gise`le Bonne, PhD,
Anders Oldfors, MD, PhD, Jocelyn Laporte, PhD, and Norma Beatriz Romero, MD, PhD
From the Neuromuscular Morphology Unit, Myology Institute, GHU Pitie- Abstract
Salp^etrie`re, Paris, France (RA -P, EM, GB, MTB, AM, CL, MB, EL,
NBR); FISEVI-UGC Anatomia Patologica-HU Virgen del Rocio, Se- Titin-related myopathies are heterogeneous clinical conditions as-
villa, Spain (RA -P); University of Granada, Granada, Spain (RA -P); Sor- sociated with mutations in TTN. To define their histopathologic
bonne University, INSERM UMRS974, GHU Pitie-Salp^etrie`re, Paris, boundaries and try to overcome the difficulty in assessing the patho-
France (IN, MB, GB, NBR); AP-HP, GHU Pitie-Salp^etrie`re, Centre de genic role of TTN variants, we performed a thorough morphological
Reference des Maladies Neuromusculaires Nord/Est/Ile de France, Paris, skeletal muscle analysis including light and electron microscopy in
France (EM, BE, EL, NBR); Department of Translational Medicine, 23 patients with different clinical phenotypes presenting pathogenic
IGBMC, INSERM U1258, UMR7104, Strasbourg University, Illkirch, autosomal dominant or autosomal recessive (AR) mutations located
France (XL, CC, JB, RS, JL); Assistance Publique-Ho^pitaux de Paris in different TTN domains. We identified a consistent pattern charac-
(AP-HP), GH Cochin-Broca-Ho^tel Dieu, Laboratoire de Biochimie et terized by diverse defects in oxidative staining with prominent nu-
Genetique Moleculaire, Paris, France (JN, FL); Department of Pathology clear internalization in congenital phenotypes (AR-CM)
and Genetics, Institute of Biomedicine, University of Gothenburg, Goth- (n � 10), 6 necrotic/regenerative fibers, associated with endomysial
enburg, Sweden (CHO, AO); Hospital Nacional de Pediatria J.P. Garra- fibrosis and rimmed vacuoles (RVs) in AR early-onset Emery-Drei-
han and Instituto de Investigaciones Neurologicas FLENI, Buenos Aires, fuss-like (AR-ED) (n � 4) and AR adult-onset distal myopathies
Argentina (SM, FL, ALT); Neuromuscular Research Center, Tampere (n � 4), and cytoplasmic bodies (CBs) as predominant finding in he-
University and University Hospital, Tampere, Finland (BU); Folkhalsan reditary myopathy with early respiratory failure (HMERF) patients
Institute of Genetics, Helsinki University, Helsinki, Finland (BU); Centre (n � 5). Ultrastructurally, the most significant abnormalities, partic-
National de Recherche en Genomique Humaine (CNRGH), Institut de ularly in AR-CM, were multiple narrow core lesions and/or clear
Biologie Franc�ois Jacob, CEA, Evry, France (AB, JFD); Genethon Insti- small areas of disorganizations affecting one or a few sarcomeres
tute, Evry, France (IR); and Complex Systems and Translational Bioin- with M-band and sometimes A-band disruption and loss of thick fil-
formatics, ICube, Strasbourg University, CNRS UMR7357, Illkirch, aments. CBs were noted in some AR-CM and associated with RVs
France (RS, JT) in HMERF and some AR-ED cases. As a whole, we described recog-
nizable histopathological patterns and structural alterations that
Send correspondence to: Norma Beatriz Romero, MD, PhD, Myology Insti- could point toward considering the pathogenicity of TTN mutations.
tute, Sorbonne University, GHU La Pitie-Salp^etrie`re, 75013 Paris,
France; E-mail: nb.romero@institut-myologie.org Key Words: Congenital myopathies, Electron microscopy, M-line
disruption, Muscle histopathology, Sarcomere disorganizations,
Rainiero A vila-Polo and Edoardo Malfatti contributed equally to this Titin, TTN-related myopathies.
work.
INTRODUCTION
This study was financially supported by Association Franc�aise contre les Titin is the largest human protein (33 000 amino acids)
Myopathies (AFM-Telethon, 20323, 21267), AIM Association Insti- (1, 2) and is encoded by the TTN gene (OMIM *188840) on
tut de Myologie, the Assistance Publique-Ho^ pitaux de Paris, the chromosome 2q31 (3�5). Titin is expressed in both skeletal
Institut National de la Sante et de la Recherche Medicale, the Sor- and cardiac muscles (6�8) and is located in the sarcomere
bonne Universite, the University of Strasbourg, the Centre National extending from the Z-disc to the M-line (9). Its amino-terminal
de la Recherche Scientifique, the Instituto de Salud Carlos III (M-
AES 2015), the Fundacion Publica Andaluza para la Gestion de la
Investigacion en Salud en Sevilla (FISEVI), the University of Gra-
nada (PhD International Mobility Programme 2014/2015), the France
Genomique National infrastructure, funded as part of the Investisse-
ments d'Avenir program managed by the Agence Nationale pour la
Recherche (ANR-10-INBS-09), and by Fondation Maladies Rares
within the frame of the "Myocapture" sequencing project, the ANR-
10-LABX-0030-INRT under the frame program Investissements
d'Avenir ANR-10-IDEX-0002-02, and the Swedish Research
Council.
The authors have no duality or conflicts of interest to declare.
VC 2018 American Association of Neuropathologists, Inc. All rights reserved. 1101
Avila-Polo et al J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018
domain is anchored to the Z-disc interacting with many pro- data of these patients were systematically retrieved and retro- Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
teins at this level (10�12). The I-band region is responsible of spectively analyzed (Table 1). Patients were classified into 4
the elastic property of the protein (13, 14) whereas the A-band groups according to their clinical features as follows: Group 1:
region represents the largest part of the protein and is a rigid Autosomal recessive congenital myopathy (AR-CM) (n � 10);
portion tightly associated to myosin, providing stabilization to Group 2: Autosomal recessive early-onset Emery-Dreifuss-
the sarcomere (5). The carboxy-terminal domain contains a ki- like myopathy without associated cardiomyopathy (AR-ED)
nase domain. At this level, titin filaments from the adjacent (n � 4); Group 3: Autosomal recessive young or early-adult
half-sarcomere overlap and connect with other protein ele- onset distal myopathy (AR-DM) (n � 4); and Group 4:
ments such as myomesin (15) and calpain 3 (16). Titin has a HMERF (n � 5). All patients underwent open biopsy for mor-
critical role in the maintenance of the sarcomere structure dur- phological and histochemical analyses of fresh-frozen skeletal
ing the contraction (17�19). muscle tissue.
Next generation sequencing (NGS) approaches have led Mutation Analysis
to an exponential increase in the number of identified muta- Patients or parents gave informed consent for the genetic
tions, either pathogenic mutations or changes of unknown sig-
nificance, in TTN. To date, mostly through NGS methods, analysis and DNA storage according to French legislation
TTN mutations have been associated with a large spectrum of (Comite de Protection des Personnes Est IV DC-2012-1693).
clinical conditions ranging from isolated dilated or hypertro- Genomic DNA was extracted from blood or frozen skeletal
phic cardiomyopathies (MIM #604145; MIM #613765) (20� muscle by standard methods. DNA was studied by direct se-
23) to numerous skeletal muscle myopathies (MIM #600334, quencing of exons of TTN gene or exome sequencing. Exome
MIM #608807, MIM #603689, MIM #611705) (24�26). How- sequencing was performed for patients P1, P2, P3, P4, P5, P7,
ever, not all rare TTN variants are associated with a disease P8, P9, P10, P17, P18, and P19 with the SureSelect Human
and in this respect a recent study by Savarese et al highlighted All Exon 50 Mb Capture Library v5, P6 and P14 with SureSe-
how challenging is the assignment of new mutations to a plau- lect Human All Exon Capture Library v6 (Agilent, Santa
sible titinopathy (27) and that, in all likelihood, new pheno- Clara, CA) and paired-end sequenced on a HiSeq 2500 (Illu-
types may emerge in the future. For this reason, mina, San Diego, CA). Confirmation of variants and segrega-
histopathological phenotype and genotype correlations are of tion was performed by Sanger sequencing of genomic DNA
critical importance, particularly because functional studies are and cDNA (Transcript variant IC, References Sequences
possible only for some mutations located in specific TTN NM_001267550.2), with standard techniques. P20 mutations
domains (28). were determined by Sanger sequencing. Sequencing
primers are available on request. PCR was performed with
Histopathological changes in TTN-related myopathies DreamTaq DNA polymerase according to standard protocol
are markedly variable as reported in muscle biopsies from (Fermentas, Waltham, MA). PCR products were sequenced on
patients with tibial muscular dystrophy ([TMD]; MIM an ABI3730xl DNA Analyzer (Applied Biosystems, Foster
#600334) and limb-girdle muscular dystrophy 2J ([LGMD2J]; City, CA), using the Big-Dye Terminator v3.1 kit and ana-
MIM #608807) with rimmed vacuoles (RVs) (29, 30), or he- lyzed with Sequencher 5.0 software (Gene Codes Corp., Ann
reditary myopathy with early respiratory failure ([HMERF]; Arbor, MI).
MIM #603689) with cytoplasmic bodies (CBs) (31�35).
Moreover, increased nuclear internalization and deficits in ox- Morphological Studies
idative staining described as minicores/minicore-like lesions Open muscle biopsy was performed for all patients after
have been reported in early-onset myopathies (28, 36, 37).
Nevertheless, to date, a systematic and exhaustive skeletal informed consent. Age at biopsy varied from 1 month (P9) to
muscle histopathologic and ultrastructural analysis have not 71 years (P20). The biopsied muscle is reported in Table 2.
been performed in large cohorts of TTN-related conditions. Samples were analyzed either in our research laboratory at the
Myology Institute in Paris, France, or at the Neuropathology
In order to define histopathologic boundaries of TTN- laboratory of FLENI-Institute and J.P. Garrahan Hospital in
related myopathies and help both clinicians and geneticists Buenos Aires, Argentina, or at the Department of Pathology,
supporting the pathogenic role of TTN variants, we report a Sahlgrenska University Hospital in Gothenburg, Sweden. For
systematic histopathological and ultrastructural analysis of 23 conventional histochemical techniques, 10-lm-thick cryostat
patients with TTN mutations presenting different clinical sections were stained with hematoxylin and eosin (H&E),
phenotypes. modified Gomori trichrome, periodic acid Schiff technique,
Oil red O, reduced nicotinamide adenine dinucleotide
MATERIALS AND METHODS dehydrogenase-tetrazolium reductase (NADH-TR), succinic
dehydrogenase, cytochrome c oxidase, menadione-nitro blue
Patients tetrazolium and adenosine triphosphatase preincubated at pH
Twenty-three patients (14 male and 9 female) of various 9.4, 4.63, 4.35. Digital photographs of each biopsy were
obtained with a Zeiss AxioCam HRc linked to a Zeiss Axio-
ethnic backgrounds were included in the present study. plan Bright Field Microscope and processed with the Axio
Patients P1 to P10, P14, and P17 to P20 (n � 15) are reported Vision 4.4 software (Zeiss, Oberkochen, Germany).
herein for the first time. Part of the clinical, pathologic or ge-
netic data from patients P11 to P13, P15, P16, and P21 to P23
(n � 8) have been previously reported (35, 38�41). Clinical
1102
TABLE 1. Clinical Phenotypes and Genetic Data J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018
Patient Sex, Onset Clinical Phenotype Family TTN Mutation (Ref Seq NM_001267550.2): Exon Inheritance Titin Reference
Affected Nucleotide Change/Isoform Modification Domain
None
Group 1 Yes1 c.2137C>T/ p.(Arg713*) 14 AR Z-line PA
P1, � M (Childhood) Congenital myopathy None c.95562G>C/ p.(Trp31854Cys) A-band PA
c.56200_56215dup/ p.(Asn18739Ilefs*2) A-band PA
None c.23444G>A/ p.(Arg7815Gln) 345 I-band
None c.79070_79071del/ p.(Tyr26357Cysfs*55) A-band PA
P2, � M, 3 years Congenital myopathy c.76502T>C/ p.(Val25501Ala) 290 AR A-band PA
None c.10045A>G/ p.(Thr3349Ala) I-band
None c.4078G>A/ p.(Gly1360Arg) 82 Z-line PA
None c.64688C>G/ p.(Pro21563Arg) A-band PA
P3, � F, Birth Congenital myopathy c.97218_97221dupTATT/ p.(Lys32408Tyrfs*2) 327 AR A-band PA
None c.35713 � 1G>A/ Intron 162 (not in N2A
None 327 � PA
isoform) A-band PA
None c.53918del/ p.(Gly17973Glufs*18) 43
None c.95867G>A/ p.(Trp31956*) A-band (38) (P1)
None c.99415A>G/ p.(Lys33139Glu) 24 A-band (38) (P2)
None c.99833dup/ p.(Val33278Serfs*2) A-band (38) (P3)
P4, � M, <1 year Congenital myopathy c.71993G>C/ p.(Arg23998Pro) 311 AR A-band PA
P5*, � M, Birth Congenital myopathy Yes2 c.107425del/ p.(Asp35809Thrfs*35) M-line
Yes3 c.97417del/ p.(Arg32473Valfs*19) 350 A-band
c.17009G>A/ p.(Trp5670*) � AR A-band
c.19715-1G>C/ splice A-band
c.29621_29624del/ p.(Glu9874Glyfs*28) 281 I-band
c.102214T>C/ p.(Trp34072Arg) M-line
P6 F, Birth Congenital myopathy, mainly distal 346 AR
c.106959T>A/ p.(Tyr35653*) (homozygous) M-line
Congenital myopathy with severe di- 356
lated cardiomyopathy (11 y) c.106050del/ p.(Glu35351Asnfs*54) M-line
P7, � M, 2 c.106978C>T/ p.(Gln35660*) 356 AR M-line
Congenital myopathy with early dif- c.105910_105914del/ p.(Thr35304Cysfs*3) M-line
fuse contractures and cardiopathy c.106422del/ p.(Phe35475Serfs*3) 327 M-line
(23 y) c.26877G>A/ p.(Trp8959*) I-band
P8*, � F, Birth c.107387A>C/ p.(Glu35796Ala) 363 (Mex5) AR M-line
Severe congenital myopathy with
arthrogrypotic features c.100558_100561dup/ p.(Gly33521Aspfs*25) 350
c.107647del/ p.(Ser35883Glnfs*10)
P9, � M, Birth Congenital myopathy with early 59 AR
P10, � M, Birth diffuse contractures c.98105del/ p.(Pro32702Leufs*15)
c.107647del/ p.(Ser35883Glnfs*10) 68
Group 2 Early-onset recessive Emery-Dreifuss-
P11*, � M, 10 like without cardiomyopathy 105 AR
P12*, � F, Childhood
P13*, � F, 19 months Early-onset recessive Emery-Drei- 359 (Mex1)
P14, � M, 20 years fuss-like without cardiomyopathy
361 (360�) (Mex2) AR
Early-onset recessive Emery-Drei-
fuss-like without cardiomyopathy 359 (358�) (Mex1) AR Titinopathies: Ultrastructural Particularities
361 (360�) (Mex3)
Young adult recessive proximal 359 (358�) (Mex1) AR
weakness with mild contractures 360 (359�) (Mex2)
without cardiomyopathy
94 AR
Young adult onset recessive distal
titinopathy 363 (Mex5)
Group 3 358 (357�) AR A-band (5, 39A)
P15* M, 20 years M-line (40) (C1)
(41) (B)
363 (362�) (Mex5) A-band M-line (5, 39B)
(40) (C2)
P16* F, 30 years Young adult onset recessive distal 353 (352�) AR
titinopathy
1103 363 (362�) (Mex5)
(continued)
Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
Avila-Polo et al Reference (41) (C) All listed variations are heterozygous and predicted to affect N2A mature skeletal muscle isoform except otherwise stated. J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018
PA *Patients are carriers of truncating mutations or of mutations previously reported.
1104 PA Electron Microscopy (EM)
PA Detailed EM analysis was performed in 19 patients
PA
(35) (L: II-1) Patients are carriers of 1 truncating mutation and 1 missense mutation found in an exon already reported as mutated in the disease. (Table 2). For ultrastructural studies, small muscle samples
(35) (K: II-1) were fixed in 2.5% glutaraldehyde, pH 7.4, postfixed in 2% os-
(35) (G: II-2) mium tetroxide for 2 hours, dehydrated and embedded in ep-
oxy resin. At least 3 blocks from each patient were studied,
Titin M-line Patients are carriers of 1 truncating mutation and 1 missense mutation found in an exon never reported as mutated in the disease. including longitudinal and transverse-oriented samples. Semi-
Domain A-band �Segregation in the family was confirmed. thin sections were stained by toluidine blue and examined in
M-line �Numbered as firstly published, according to the old numbering (before October 2013); AR: Autosomal recessive; AD: Autosomal dominant. light microscopy to select pathological areas. Ultrathin sec-
A-band **Described in the original paper as semirecessive or semidominant (34); M: Male; F: Female; Ref Seq: Reference Sequence; PA: Present Article; HMERF: Hereditary Myopathy with Early Respiratory Failure; 1: sisters; 2: his tions were stained with uranyl acetate and lead citrate. The
A-band mother (P16); 3: her son (P15); 4: his sister (P18); 5: her brother (P17); 6: her father; 7: her brother; 8: his mother. grids were observed using a Philips CM120 electron micro-
A-band scope (80 kV; Philips Electronics NV, Eindhoven, The
A-band Netherlands).
A-band
A-band RESULTS
Inheritance 364 (Mex6) AR AD AD ** ** AD Clinical Findings Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
274 Clinical summary and laboratory features of all patients
Exon 344 344 344 (343�) 344 (343�) 344 (343�)
364 (Mex6) AR are provided in Table 1.
274 Group 1: AR-CM patients, P1�P10, showed congenital
Family TTN Mutation (Ref Seq NM_001267550.2): c.107867del/ p.(Leu35956Argfs*16) hypotonia or early onset diffuse muscle weakness, with con-
Affected Nucleotide Change/Isoform Modification c.52021C>T/ p.(Arg17341*) genital arthrogryposis or early development of contractures
c.107867del/ p.(Leu35956Argfs*16) (P8, P9, and P10); associated cardiomyopathy was found in P7
c.52021C>T/p.(Arg17341*) and P8.
c.95185T>C/ p.(Trp31729Arg)
c.95187G>C/ p.(Trp31729Cys) Group 2: AR-ED patients, P11�P13 have been recently
c.95195C>T/ p.(Pro31732Leu) reported (38) and P14 is firstly described in this report.
c.95195C>T/ p.(Pro31732Leu) (homozygous)
c.95134T>C/ p.(Cys31712Arg) Group 3: AR-DM patients, P15�P18, showed distal or
proximal-distal early severe muscle weakness of the 4 limbs.
Yes4 P15 and P16 were described elsewhere (39, 41). P17 and P18
Yes5 are reported here for the first time.
Yes6
None Group 4: HMERF group included patients P19�P23.
Yes7 Most of them had adult onset weakness with diaphragmatic re-
None spiratory failure. Earlier onset (10 years) has been observed in
Yes8 P19. Patients P21�P23 were previously reported (35).
Clinical Phenotype Young adult recessive proximal & Molecular Data
distal myopathy To identify the mutations in patients without a genetic
Young adult recessive proximal & diagnosis, we performed exome sequencing on genomic DNA
distal myopathy from the patients and their parents. Exome sequencing allows
a fast and parallel screening of most human genes, and is suit-
HMERF able and efficient for the diagnosis of neuromuscular diseases
HMERF and the analysis of large genes such as TTN (42). This ap-
HMERF proach also covers any newly discovered gene for the
HMERF disorder.
HMERF
For all patients, we found known or novel mutations in
TABLE 1. Continued Patient Sex, Onset P17*, � M, 20 years TTN (Table 1). These changes were confirmed by Sanger se-
P18*, � F, 30 years quencing, and their familial segregation validated when paren-
Group 4 tal DNA was available. Parents of patients P1, P2, P3, P4, P5,
P19* F, 10 years P7, P8, P9, P10, P11, P12, P13, P14, P17, and P18 were
P20* M, 50 years screened to confirm the segregation of the mutations and to
P21* F, 54 years verify that TTN mutations are on opposite alleles (in trans po-
P22* M, 25 years sition). Most of the patients harbored compound heterozygous
P23* M, 38 years mutations (21 patients), at least one of them was a truncating
mutation (frameshift, nonsense mutation or mutation affecting
an essential splice site) leading to a predicted protein trunca-
tion or degradation (Table 1). The second mutations were
either truncating mutations or rare missense mutations.
Two cases carried homozygous mutations: P11 (AR-ED) and
J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018 Titinopathies: Ultrastructural Particularities
TABLE 2. Patient Data and Summarized Histological Findings
Patient Age at Biopsy Muscle Biopsied Method Light Microscopy Electron Microscopy Reference
Group 1
P1 8 y; 13 y NR LM, EM Well-defined areas of defective oxi- Focal and short areas of myofibrillar PA
Vastus lateralis
Deltoid dative staining, some nuclear disorganization, nuclear internal-
Vastus lateralis
Deltoid internalizations, type 1 fiber izations; some clear areas with
Deltoid
Deltoid predominance myofilaments loss, M-line disrup-
Deltoid
Vastus lateralis tion and almost intact Z-line
Vastus lateralis
P2 7y LM, EM Multiple and well-delimited areas of Focal and multiple areas of myofi- PA
uneven oxidative staining, central- brillar disarray resembling mini-
ized nuclei, type 1 fiber cores, nuclear internalizations
predominance Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
P3 3y LM, EM Multiple and well-delimited areas of Foci of myofibrillar disorganization PA
uneven oxidative staining, promi- with Z-band streaming running
nent nuclear centralizations, type 1 over few sarcomeres, some large
fiber uniformity clear areas with myofilaments
loss, M-line disruption and almost
intact Z-line
P4 7y LM, EM Type 1 fiber predominance, internal- Focal and multiple areas of PA
ized nuclei, uneven oxidative myofibrillar disarray resembling
staining minicores, nuclear internaliza-
tions, Z-line streaming spanning
all along the sarcomere resembling
"pennants"
P5 31 y LM, EM Uneven oxidative staining, numerous Z-line streaming spanning all along PA
internalized nuclei, type 1 fiber the sarcomere or as small
predominance "pennants", rare focal areas of
myofibrillar disorganization
P6 49 y LM, EM Type 1 fiber predominance, numer- Z-line streaming spanning all along PA
ous internalized nuclei, uneven ox- the sarcomere or as small
idative staining, increased "pennants", internalized nuclei in
interstitial fat and connective tis- rows, small RVs, focal areas of
sue, occasional COX negative myofibrillar disorganization
fibers.
P7 12 y LM, EM Multiple and well-delimited areas of Focal and multiple areas of myofi- PA
uneven oxidative staining, promi- brillar disarray resembling mini-
nent nuclear centralizations, type 1 cores, granular streaming spanning
fiber uniformity all along the sarcomere or as small
"pennants", internalized nuclei
P8 23 y LM, EM Prominent nuclear centralizations, Z-line streaming spanning along the PA
type 1 fiber uniformity, presence sarcomere or as small "pennants",
of rare areas of uneven oxidative internalized nuclei
staining
P9 1m LM, EM Fiber caliber variation, many small Focal and multiple areas of myofi- PA
fibers, internalized nuclei, uneven brillar disarray resembling mini-
oxidative staining, fuchsinophilic cores, clear areas with loss of
protein inclusions myofilaments with almost intact
Z-line, CBs, numerous fibers with
marked sarcomere disorganization
P10 4 m; 4 y LM, EM Fuchsinophilic protein inclusions, CBs, nuclear filamentous inclusions, PA
uneven oxidative staining, nuclear atrophic fibers with degenerated
internalizations myofibrils, duplication of triads
(continued)
1105
Avila-Polo et al J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018
TABLE 2. Continued
Patient Age at Biopsy Muscle Biopsied Method Light Microscopy Electron Microscopy Reference
Group 2
P11 7y Deltoid LM, EM Necrotic/regenerating fibers, some Clear areas with loss of myofila- (38) (P1)
Deltoid LM, EM nuclear internalizations, and type ments with almost intact Z-line, (38) (P2)
P12 43 y; 52 y 1 fiber predominance internalized nuclei
Vastus lateralis LM, EM (38) (P3)
P13 9y Rare necrotic/regenerating fibers, CBs, sometimes with dense material Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
Deltoid LM, EM nemaline bodies, RVs, uneven ox- in the peripheral halo, nemaline PA
P14 47 y idative staining, and some nuclear bodies, nuclear tubulo-filamentous
Deltoid internalizations inclusions, RVs/AV, clear areas
with loss of myofilaments with al-
Deltoid Type 1 fiber predominance, rods, most intact Z-line, internalized
Deltoid RVs, uneven oxidative staining, nuclei
NR internalized nuclei, necrotic and
Deltoid regenerative fibers CBs, sometimes with dense material
in the peripheral halo, nemaline
Uneven oxidative staining, internal- bodies, tubulo-filamentous mate-
ized nuclei, increased interstitial rial, RVs, and filamentous nuclear
connective tissue, occasional COX inclusions, focal Z-line streaming
negative fibers spanning all along the sarcomere
or as small "pennants".
Z-line streaming spanning all along
the sarcomere or as small
"pennants", mini-cores, internal-
ized nuclei, nemaline rods in some
fibers, mitochondrial
abnormalities
Group 3
P15 42 y LM, EM Uneven oxidative staining, internal- Clear areas with loss of myofila- (39) (5A)
ized nuclei ments with almost intact Z-line, Z- (40) (C1)
P16 69 y LM, EM line streaming spanning all along (41) (B)
LM Uneven oxidative staining, internal- the sarcomere or as small
P17 54 y LM ized nuclei "pennants", large areas of protein (39) (5B)
material and myofibrillar loss, in- (40) (C2)
Necrotic fibers, few RVs, internal- ternalized nuclei (41) (C)
ized nuclei, mild uneven oxidative
staining, Type 1 fiber predomi- Z-line streaming spanning all along PA
nance, mild endomysial fibrosis the sarcomere or as small
"pennants", large areas of accumu-
Few internalized nuclei (only H&E lated protein material with myofi-
staining available) brillar loss.
NA
P18 NS NA PA
Group 4
P19 10 y LM, EM CBs, uneven oxidative staining, in- CBs sometimes with dense protein PA
ternalized nuclei aggregates in the peripheral halo,
RVs, Z-line streaming as small
"pennants", large areas of protein
material and myofibrillar loss,
atrophic fibers with disorganized
internal structure with thin-fila-
ments and small segments of
dense material
(continued)
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TABLE 2. Continued
Patient Age at Biopsy Muscle Biopsied Method Light Microscopy Electron Microscopy Reference
P20 71 y Peroneus longus LM Rods, CBs fuchsinophilic protein NA PA
inclusions, slight increase in endo-
mysial fibrosis, type 1 fiber pre-
dominance, internalized nuclei
P21 55 y Deltoid LM, EM Type 1 fiber predominance, CBs, CBs sometimes with dense material (35) (L: II-1)
RVs, uneven oxidative staining, in peripheral halo, protein inclu-
internalized nuclei sions, RVs, large areas of protein
material and myofibrillar loss, in-
ternalized nuclei
P22 27 y Deltoid LM CBs, RVs, uneven oxidative staining, NA (35) (K: II-1) Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
internalized nuclei
P23 48 y Deltoid LM EM CBs, RVs, regenerative fibers, inter- CBs, large areas of protein material (35) (G: II-2)
nalized nuclei and myofibrillar loss, atrophic
fibers with disorganized internal
structure with thin-filaments and
small segments of dense material
Abbreviations: y: years; m: months; NR: not referred; LM: light microscopy; EM: electron microscopy; PA: present article; CBs: cytoplasmic bodies; RVs: rimmed vacuoles;
NA: not available; H&E: hematoxylin and eosin.
FIGURE 1. Histochemistry from group 1 patients (AR-CM). P3 (A�C): (A) H&E. Presence of numerous fibers harboring
centralized nuclei. (B) NADH. The majority of muscle fibers show multiple and irregular areas of defective oxidative reaction. (C)
ATPase 9.4. Type 1 fiber predominance. P5 (D�F): (D) H&E. Great fiber size diameter variation and presence of multiple nuclear
internalization in numerous fibers. (E) NADH. Multiple and irregular centrally placed areas of defective oxidative reaction. (F)
ATPase 9.4. Type 1 fiber predominance.
P22 (HMERF). Several mutations were previously known or the protein, they mostly were located within the A-band (9
affected exons already mutated in the disease. The muta- of 10 cases). I-band was involved in 3 of 10 cases, and both
tions were located in different protein domains (Table 1). M-line and Z-line in 2 of 10 cases, respectively. As recently
Patients from Group 1 (AR-CM) had the most heteroge- reported, patients P11�P13 from Group 2 (AR-ED) had TTN
neous results. Although variants were distributed all along mutations located in exons coding for the M-line domain
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Avila-Polo et al J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018
FIGURE 2. Electron microscopy studies from Group 1 patients (AR-CM). (A, B) P3. Characteristic lesions showing evident loss of Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
thick myofilaments with M-line and A-line dissolution and slight fragmentation of Z-line involving a few or several sarcomeres
and myofibrils. (C, D) P5 and P7. Small areas of myofibrillar disarray involving one or few sarcomeres with dispersion of Z-line
material resembling small "pennants". (E) P1. Focal and large area of myofibrillar disorganization with Z-line streaming and
paucity of mitochondria. (F) P1. Small area of sarcomeric disarray with accumulation of Z-line material spanning one sarcomere.
Areas of myofibrillar disorganization extended through the adjacent myofibrils.
(Mex1�Mex3) (38). P14 showed a similar phenotype with different TTN exons coding for M-line domain (Mex) had
severe proximal weakness mainly in lower limbs and limita- calpain deficiency without mutations in CAPN3.
tion in abduction of the arms due to mild contracture of the
shoulder, but mutations were located in exons 363/Mex5 Morphological Findings
and 94 (I-band). In Group 3 (AR-DM), a combination of Detailed histopathologic analysis is reported in Table 2.
Mex5/Mex6 mutations and a second mutation involving Group 1: AR-CM (P1-P10) (Figs. 1 and 2). Muscle biop-
A-band domain was present in all patients (39�41). Finally,
HMERF patients carried several reported mutations in exon sies from all cases showed multiple small irregular and ran-
344 (35). We studied Calpaine 3 on Western blot in 5 domly distributed areas of reduced/absent oxidative activity or
patients (P6, P11, P12, P13, and P14). Four patients (P11, better-defined core areas (P1, P2, P3, P7) associated with mild
P12, P13, and P14) harboring at least one mutation in fiber size variability, type 1 fiber predominance and increased
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J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018 Titinopathies: Ultrastructural Particularities
FIGURE 3. Histochemistry from Group 2 (AR-ED) and Group 3 patients (AR-DM). (A�C) P13 (AR-ED): (A) H&E. Presence of great Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
round fibers size variation with numerous atrophic fibers, some necrotic fibers, rimmed vacuoles (arrow), internalized nuclei and
endomysial fibrosis. (B) NADH. Numerous fibers showing diffuse alterations of oxidative staining conferring a lobulated aspect
to smaller fibers. (C) Type 1 fiber predominance. (D�F) P17 (AR-DM). (D) H&E. Fibers type variation, prominent nuclear
internalizations and mild endomysial fibrosis, necrotic/regenerative fibers (arrow). Presence of some rimmed vacuoles (GT in top
right corner). (E) NADH. Irregular areas of uneven oxidative staining. (F) ATPase 9.4. Marked type 1 fiber predominance.
nuclear internalizations (Fig. 1A�F). Increased frequency of most of the cases. Ultrastructural study showed also the pres-
nuclear centralizations was evident in 4 cases (P2, P3, P7, P8). ence of clear small and focal areas of sarcomere M-line disso-
In addition, P9 and P10 displayed numerous CBs in atrophic lution spanning one or a few sarcomeres with almost complete
fibers. Using EM, we identified variable length sarcomere preservation of the Z-line (P11, P12) (Fig. 4A, B), and dense
disorganizations characterized by (i) clear small areas of Z-line material disrupted (Fig. 4C), or resembling small
M-line dissolution by subsequent disintegration of thick fila- "pennants" (P13, P14). CBs were observed in P12 and P13;
ments, running along one or a few sarcomeres with preserva- some of them showed small segments of dense material
tion of the Z-line structure (P1, P3, P7, P9) (Fig. 2A, B); some among the thin filaments in the peripheral halo. Moreover,
areas of myofibrillar disorganization extended through the ad- nemaline bodies (P12, P13, P14) with the characteristic square
jacent myofibrils; (ii) focal areas of diffusion of Z-line mate- lattice structure (Fig. 4D), RVs containing degradation prod-
rial resembling small "pennants" starting from the Z-line (P4, ucts (P13, P14), tubulofilamentous sarcoplasmic (P13) and nu-
P5, P6, P7, P8) (Fig. 2C, D); sometimes, the electron dense clear inclusions (P12, P13) were also observed.
material appeared to span the full width of a sarcomere
(Fig. 2F); (iii) many focal areas of sarcomeric disruption af- Group 3: AR-DM (P15�P18) (Figs. 3D�F, 5A�C). Mus-
fecting a few sarcomeres with Z-line streaming and sharp lim- cle biopsies showed mild irregular disorganizations at oxida-
its from the adjoining normal sarcomeres with paucity of tive staining associated with mild to moderate myopathic
mitochondria, evoking classical minicores (P1, P2, P3, P4, P6, changes (Fig. 2D�F). Few necrotic and regenerative fibers, as
P7, P9) (Fig. 2E). Moreover, P9 and P10 showed additional well as RVs in sparse fibers were evident in P17. By EM, we
changes as CBs (P9, P10), nuclear inclusions corresponding to showed the presence of small and focal areas involving one
tubulofilamentous aggregates (P10), duplication of triads sarcomere as clear areas with loss of myofilaments including
(P10) or atrophic fibers showing disorganized internal struc- M-line dissolution with almost intact Z-line (P15) (Fig. 5A),
ture with thin filaments and small segments of dense Z mate- Z-line diffusion spanning all along the sarcomere (Fig. 5B) or
rial (P9, P10). Internalized/centralized nuclei were common in resembling small "pennants" (P15, P16), or large areas of ac-
most of the cases. cumulated dark protein material and myofibrillar striation loss
(P15, P16) (Fig. 5C).
Group 2: AR-ED (P11�P14) (Figs. 3A�C, 4). Muscle bi-
opsies showed a dystrophic pattern with marked fiber size var- Group 4: HMERF (P19�P23) (Figs. 5D�F, 6). All biop-
iability, necrotic and regenerative fibers, endomysial fibrosis, sies showed internalized nuclei (Fig. 6A), and numerous fuch-
numerous RVs and sometimes rods or CBs (Fig. 3A�C). In ad- sinophilic inclusions corresponding to CBs, presenting a
dition, irregularities in oxidative staining were also evident in remarkable circular disposition in some fibers in P19 and P21
(Fig. 6B�D). By EM some CBs showed small electron dense
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Avila-Polo et al J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018
FIGURE 4. Electron microscopy from Group 2 (AR-ED) (A) P12. Distinctive lesion showing loss of myofilaments with M-line Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
dissolution, disintegration of thick filament and almost preserved Z-line, involving a few sarcomeres and myofibrils; (B) Clear
areas of myofibrillar disintegration extending to the adjacent sarcomeres corresponding to dissolution of M-band structure, with
almost intact Z-line. (C) P14. Small areas of disorganization of the structure with Z-line material accumulation occasionally
reminding a classical minicore. (D) P12. Cytoplasmic elongated nemaline bodies (rods), with the characteristic square lattice
structure (in top right corner).
amorphous inclusions among the thin filaments in the periph- scaffolding as common histopathologic lesions associated
eral halo (P19 and P21) (Fig. 5D, E). Widespread abnormal with TTN pathogenic mutations.
areas corresponding to accumulated filamentous material and
damage of myofibrils with loss of striations were frequently DISCUSSION
observed (P19, P21, P23). Furthermore, we rarely found focal TTN-related myopathies comprise a large group of dif-
sarcomere disruptions with small "pennants" starting from the ferent clinical entities (24�26). The relatively recent employ-
Z-line (P19, P23). Eventually, we encountered some atrophic ment of NGS techniques has led and probably will continue to
fibers with completely disorganized internal structure and rods lead to increased numbers of genetic variants described in
(P19), and rimmed/autophagic vacuoles (P19, P21, P23) TTN as well as new associated clinical phenotypes (28, 38, 43,
(Fig. 5F). 44). Our study focuses on the description of recessive TTN
cases and HMERF cases. Interestingly, while HMERF is usu-
Summary ally a dominant disorder, P22 presented with homozygous
Overall, Group 1 (AR-CM) was characterized by multi- p.Pro31732Leu mutation. This mutation (initially described as
p.Pro30091Leu) was reported by Palmio et al in 2014 as a
ple and small particular areas of sarcomere disorganization semirecessive mutation since some heterozygous carriers
distributed through the muscle fibers. In AR-ED and AR-DM, don't develop a disease and some do (37). An outstanding di-
sarcomere disruptions were associated with mild myopathic agnostic challenge is the assignment of a pathogenic value to
changes or moderate dystrophic pattern 6 RVs. In HMERF the already huge and still increasing number of TTN sequence
patients, CBs were the main feature, with a typical but not variants identified with NGS (27). Moreover, reliable func-
constant circular peripheral distribution. tional tests for the interpretation of single variants are lacking.
However, considering the high number of variants of uncertain
As a whole, in all muscle biopsies we identified variable significance in the TTN gene, confrontation of molecular,
sarcomere disruptions suggesting a loss of sarcomeric
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J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018 Titinopathies: Ultrastructural Particularities
FIGURE 5. Electron microscopy studies from Group 3 (AR-DM). (A) P15. Loss of myofilaments with M-line dissolution (in the Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
central area of the image) and preserved Z-line involving one sarcomere; and in a small part are noted reminiscent of M-band.
(B) P16. Streaming of dark material starting from the Z-line and spanning all along the sarcomere; (C) P15. Large areas of
accumulated filamentous/granular material and myofibrillar striation loss. EM studies from Group 4 (HMERF). (D) P21. Muscle
fiber with subsarcolemmal cytoplasmic bodies disposed in a circle. (E) P21. Compact central area of a cytoplasmic body
harboring small electron-dense structures in the peripheral region. (F) P19. Autophagic material and cellular debris.
clinical and morphological data is crucial for the establishment disclosing the presence of distinctive small and focal areas of
of the molecular diagnosis. sarcomere disorganizations with M-line dissolution in all
patients (Fig. 4A, B). Some of CBs had an atypical aspect with
With the morphological studies of 23 TTN mutated small dense material in the peripheral halo (Table 2).
patients, we intended to correlate specific histopathological
lesions with each different clinical group of patients. AR-CM Group 3 patients P15 and P16 were originally consid-
patients (P1�P10) from Group 1 presented a typical congenital ered as severe early-onset TMD with proximal involvement
myopathy phenotype (Table 1). Cardiomyopathy was present (39), and shared a mutation in exon 363/Mex5 but harbored
in 2 cases (P7 and P8) and was reported in a clinically affected different second truncating mutations that explained their
sister of P2. All patients harbored recessive compound hetero- more severe phenotype (39�41) allowing them to be redefined
zygous mutations in different exons of TTN involving different as AR-DM. P17 and P18 harbored the same 2 mutations in
domains but predominantly located in the A-band. Muscle bi- exons 364/Mex6 and 274. Muscle biopsies revealed multiple
opsies showed disorganizations of the mesh of the intermyofi- nuclear internalization and uneven oxidative staining. Mild
brillar network at oxidative staining as predominant findings, variability in the size of the fibers, slight increase in endomy-
associated with type 1 fiber predominance, internalized or cen- sial connective tissue and some RVs were evident in P17. EM
tralized nuclei, corresponding to some extent to previously de- mainly showed both focal and large areas of disorganizations
scribed pathological spectrum of the disorder (28, 36, 37). with myofibrillar/filaments loss (Fig. 5A�C) (P15 and P16).
Nevertheless, ultrastructural studies (Table 2) consistently
revealed common and relevant features: (1) small clear areas of Group 4 included HMERF patients (P19�P23) charac-
focal myofibrillar disintegration with loss of thick filament cor- terized by severe early respiratory insufficiency with variable
responding to dissolution of M-band structure, and sometimes degree of muscular involvement. P19 showed uncommon
also A-band, with almost intact Z-line (Figs. 2A, B, 7A, B); (2) early-onset at 10 years old. All the patients harbored missense
areas with Z-line diffusion spanning all along the sarcomere, or TTN mutations in the same TTN exon 344 (35). Presence of
resembling small "pennants" (Fig. 2C, D, F); and (3) focal CBs was the common and most prominent histological feature
myofibrillar disorganizations with Z-line streaming and paucity (Fig. 6). CBs were preferentially subsarcolemmal, sometimes
of mitochondria, involving a few sarcomeres (Fig. 2E). with a circular peripheral distribution of CBs as described
(45). EM disclosed that some of them harbored the presence
Patients from Group 2 with AR-ED (P11�P14) harbored of short segments of dense material in the peripheral halo
compound heterozygous (P12, P13, P14) or homozygous (Fig. 5D, E). Variable length of sarcomere disorganizations
(P11) mutations involving M-line protein domains. Muscle bi- has also been reported in biopsies from HMERF patients (34,
opsies had a variably severe dystrophic pattern with RVs, rods 45�47). Our cases showed mainly large areas of protein
and CBs. Our detailed ultrastructural analysis allowed material deposit (P19, P21, P23) and Z-line streaming
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Avila-Polo et al J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018
FIGURE 6. Histochemistry from P21, Group 4 (HMERF). (A) H&E. Fibers size variability and internalized nuclei. Presence of fibers Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
with cytoplasmic bodies. (B) Modified Gomori trichrome. Fibers show numerous and multiple fuchsinophilic rounded inclusions
corresponding to cytoplasmic bodies. (C) NADH. Areas of defective oxidative staining corresponding to the zone occupied by
the cytoplasmic bodies with particular circular and peripheral distribution. (D) ATPase 9.4 Some fibers harbor areas devoid of
reactions corresponding to the cytoplasmic bodies.
resembling small "pennants" (P19, P23). Atrophic fibers Of note, these restricted and focal sarcomere abnormali-
showed complete sarcomere disorganization, rods and ties identified in TTN-related myopathies appear different
rimmed/autophagic vacuoles (P19, P21). from classical cores or minicores lesions as observed in myop-
athies related to RYR1 (OMIM *180901) and SEPN1 (OMIM
As a whole, we identified here that TTN pathogenic *606210) genes. Indeed, ultrastructurally, typical cores found
mutations cause a large spectrum of histopathologic lesions in RYR1 mutated patients correspond to wide areas of com-
always associated with particular sarcomere disruptions. Al- pacted and disorganized myofibrils, with Z-line streaming and
though one could imagine that the presence of sarcomeric pa- absence of mitochondria extending over numerous sarcomeres
thology is as an expected finding related to the loss of titin or almost along the full length of the fibers; they are sharply
intrinsic properties, the above mentioned particular sarcomeric demarcated from the normally structured zones of the muscle
disruption presented common elements such as focal myofi- fibers (48). However, mostly in RYR1 recessives, the core
brillar disintegration with loss of thick filament corresponding areas frequently occupied the whole myofiber cross sectional
to dissolution of M-band structure, with almost intact Z-line, and extended to a moderate number of sarcomeres in length
that we have never observed in the EM analyses of muscle bi- (49). In contrast, the classical minicores as found in SEPN1-re-
opsies from other congenital diseases. The sarcomere disrup- lated myopathies have poorly defined borders and are charac-
tion observed initially on the band M as shown in Figure 7A� terized by the presence of multiple foci of myofilamentary
D, then in Figures 2B and 5A, can be extended later on the disorganization, with Z-line streaming running over a few sar-
whole of the sarcomere structure, as shown in Figures 2A and comeres, even if occasionally they are longer; mitochondria
4A. The dissolution of M line with almost intact Z line could are absent from the altered areas (48). Additionally, and this is
be considered as an early lesion (Fig. 7) and, clearly, there a key point in our work, these "minicore" lesions never appear
may be different degrees of disruption of the sarcomere. Thus, as focal clear areas of myofibrillar disruption involving one or
these lesions might be considered as a common and priming few sarcomeres, with M-line disintegration and some loss of
myofibril damage that successively lead to the development of filaments in the central part of the abnormal areas associating
multiple and variable histopathological alterations.
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J Neuropathol Exp Neurol � Volume 77, Number 12, December 2018 Titinopathies: Ultrastructural Particularities
FIGURE 7. High-magnification electron microscopy studies from P3 (AR-CM) (A, B), and P15 (AR-DM) (B, C). These images Downloaded from https://academic.oup.com/jnen/article/77/12/1101/5144759 by guest on 02 August 2021
show slight abnormalities, only identified by especially M-band disruption, without A-band alterations, which could be
considered as initial or early changes.
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