Arq Neuropsiquiatr 2009;67(3-B):886-891
Clinical, histochemical and immunohistochemical features
Nazah Cherif Mohamad Youssef, Rosana Herminia Scola, Paulo José Lorenzoni, Lineu César Werneck
abstract – Nemaline myopathy (NM) is a congenital disease that leads to hypotonia and feeding difficulties
in neonates. Some cases have a more benign course, with skeletal abnormalities later in life. We analyzed a
series of eight patients with NM obtained from a retrospective analysis of 4300 muscle biopsies. Patients were
classified as having the typical form in five cases, intermediate form in two cases and severe form in one case.
Histochemical analysis showed mixed rods distribution in all cases and predominance of type I fibers in five
cases. Immunohistochemical analysis showed abnormal nebulin expression in all patients (four heterogeneous
and four absent), homogeneous desmin expression in four cases, strongly positive in three and absent in one,
fast myosin expression in a mosaic pattern in six cases and absent in two cases. There was no specific relation
between these protein expression patterns and the clinical forms of NM.
KEY WORDS: nemaline myopathy, nebulin, desmin, myosin, immunohistochemistry.
miopatia nemalínica: achados clínicos, histoquímicos e imuno-histoquímicos
Resumo – Miopatia nemalínica (NM) é uma doença congênita que leva a hipotonia e dificuldade de sugar em
neonatos. Alguns casos possuem uma evolução benigna, com deformidades ósseas tardias. Nós analisamos uma
série de oito pacientes com NM obtidos da análise retrospectiva de 4300 biópsias musculares. Os pacientes
foram classificados como forma típica em cinco casos, forma intermediária em dois casos e forma severa em
um caso. Análise histoquímica mostrou distribuição mista dos rods em todos os casos e predominância de
fibras tipo I em cinco casos. Análise imuno-histoquímica mostrou expressão anormal da nebulina em todos
os pacientes (quatro heterogênea e quatro ausente), expressão homogenea da desmina em quatro casos,
fortemente positiva em tres e ausente em um, expressão da miosina (rápida) com padrão em mosaico em
seis casos e ausente em dois casos. Não há relação específica entre a expressão destas proteínas e as formas
clínicas da NM.
PALAVRAS-CHAVE: miopatia nemalínica, nebulina, desmina, miosina, imuno-histoquímica.
Neuromuscular Disorders Service, Neurology Division, Internal Medicine Department, Hospital de Clínicas da Universidade Federal do Paraná (UFPR),
Curitiba PR, Brasil.
Received 20 February 2009, received in final form 1 July 2009. Accepted 17 July 2009.
Dra. Rosana Herminia Scola – Serviço de Doenças Neuromusculares / Hospital de Clínicas da UFPR – Rua General Carneiro 181 / 3o andar - 80060-900
Curitiba PR - Brasil. E-mail: firstname.lastname@example.org
Nemaline myopathy (NM) is a congenital myopathy
first described in 1963 by Shy and Conen, characterized
by hypotonia and general muscle weakness predominant-
ly in facial muscles, cervical and trunk flexor muscles, foot
dorsiflexor muscles and finger extensor muscles1,2. Patients
may also exhibit dysphagia, respiratory insufficiency, foot
deformities, arch palate, scoliosis, chest deformities and
superior and inferior limbs contractures1-7. Currently, NM
is classified into six different forms: severe congenital,
typical, intermediate congenital, juvenile, adult and oth-
er forms associated with cardiomyopathy, ophthalmople-
gia and the presence of intranuclear nemaline bodies on
muscle biopsy8. Adult forms are known as sporadic late-
onset nemaline myopathies (SLONM) and are associated
with HIV and monoclonal gammopathy9,10.
An NM diagnosis is made with a muscle biopsy in
which modified Gomori-trichrome (MGT) staining shows
the presence of nemaline bodies in muscle fibers in the
subsarcolemmal or intermyofibrilar region1,2,5,11,12. The his-
tochemistry also reveals features in NM such as a pre-
dominance of type I fibers and an increase in acid phos-
phatase activity5,12-14. An immunohistochemical study of
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Youssef et al.
muscle proteins shows the nature of the structural fail-
ures in NM. Several studies have shown negative and posi-
tive expression of nebulin, myosin, desmin, titin and e dys-
trophin in muscle fiber and rods5,15-18. Nemaline myopa-
thy remains genetically heterogeneous. To date, defects
in six distinct genes have been identified in cases of NM.
These include mutations in α-tropomyosin (TPM3), beta-
tropomyosin (TPM2), nebulin (NEB), α-actin (ACTA1), co-
filin2 (CFL2) and troponin T (TNNT1)4,17-28.
This study describes the clinical manifestations of ne-
maline myopathy using histochemical analysis along with
expression analysis of nebulin, desmin and myosin pro-
teins with immunohistochemistry.
A retrospective analysis of 4300 muscle biopsies performed
between January 1978 and July 2007 identified 15 patients with a
diagnosis of NM, but seven cases were excluded because there
was not enough material from the muscle biopsy for immuno-
histochemical analysis. Relevant data, including the clinical eval-
uation, serum enzymes levels, needle electromyography (EMG),
and histological-immunohistochemical aspects of the each mus-
cle biopsy, were collected. Consent to analyze the muscle biop-
sies was obtained in the out-patient clinic or during hospital ad-
mission for diagnostic investigation.
Patients were classified as typical form, intermediate form
or severe form, according to criteria established by the Inter-
national Nemalin Myopathy Consortium8. Particular attention
was paid to obstetrical history, pregnancy complications, mus-
cle weakness and tonus, breastfeeding difficulties, respiratory
failures, dysphagia, and facial and skeletal deformities (dysmor-
phisms). Other relevant data were also collected including age,
gender, course of disease and family history.
The levels of creatine kinase (CK), aldolase (AL) and lactate
dehydrogenase (LDH) were recorded as a proportion reflecting
their increase above normal limits.
The electromyography (EMG) pattern was classified as nor-
mal, myopathic, denervation or mixed (myopathic with dener-
vation findings) according to standard procedures29.
Muscle biopsies were frozen in liquid nitrogen at –170oC and
cryostat sections were stained histologically, according to stan-
dard procedures7,11. The predominance of type I fibers with an AT-
Pase reaction (pH 9.4, 4.6 and 4.3), moth-eaten fibers with NADH-
tetrazolium redutase, focal increases in acid phosphatase, and
the frequency and distribution (subsarcolemmal, intermyofibrilar
or mixed) of fibers with rods on MGT were determined by count-
ing approximately 300 to 500 muscle fibers of each specimen.
The cryostat sections (4 um) of muscle biopsies were used
for immunohistochemistry with specific primary antibodies and
secondary antibody (anti-IgG) conjugated to fluorescein for neb-
ulin, desmin and myosin proteins, according to standard proce-
dures. The primary antibodies used were an anti-nebulin mono-
clonal antibody (Sigma, dilution 1:50), an anti-desmin monoclonal
antibody (Sigma, dilution 1:20) and a fast anti-myosin monoclo-
nal antibody (Sigma, dilution 1:10). The secondary antibody con-
jugated to fluorescein was used at a dilution 30:500. Afterwards,
an immunofluorescence analysis was performed using an epif-
luorescence microscope. The immunofluorescent classification
was modified based on the descriptions of Gurgel-Giannetti et
al.15 and Imoto et al.16: (1) nebulin: absent expression, heteroge-
neous expression (diffused imperfection), or homogeneous ex-
pression; (2) desmin: absent expression, homogeneous expres-
sion, or strongly positive expression; and (3) fast myosin: absent
expression or mosaic-type expression.
There were eight patients (six female and two male)
aged 2 months to 6 years, with a mean age of 22 months.
The patients were classified as having the typical form in
five cases, the intermediate form in two cases and severe
form in one case. Muscle hypotonia since birth was pres-
ent in all cases. The other clinical findings reported were
skeletal or facial dysmorphisms, breastfeeding difficulties
and respiratory failure (Table). Dysphagia, cardiomyopa-
thy and ophtalmoplegia were not observed. No obstetric
abnormalities were observed in six cases, but gestational
complications such as a decreased fetal movements, oli-
gohydramnios and premature birth were observed in two
cases (intermediated form and severe form). No family
history was observed (Table).
Only one case (case 4) had serum CK and LDH levels
increased above the normal limits (Table).
The EMG pattern was observed in seven patients and
was myopathic in two cases, neurogenic in three cases
and normal in the remainder. The EMG revealed abnor-
malities in four cases of the typical form, two of the in-
termediate form and in the one of the severe congeni-
tal case (Table).
A muscle histological analysis was performed in all
cases and the most common abnormalities found in mus-
cle biopsies were rods in all cases, type 1 fiber predom-
inance in five cases, moth-eaten fibers in four cases and
a focal increase in fibers of acid phosphatase activity in
four cases (Fig 1 / Table). A frequency of rods above 50%
with MGT staining was found in two patients, while a fre-
quency below 50% was found in six patients (Table). The
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Youssef et al.
Fig 1. Hematoxilin-Eosin (HE) stain showing variation in fiber sizes with areas of small groups of atro-
phic muscle fibers [A]. Some of the subsarcolemmal rods are seen with HE (arrow), but become more
evident when stained with modified Gomori trichrome (MGT) (arrow) [B and C] The predominant sub-
sarcolemmal (D), intermyofibrilar [E] or mixed rods [F and G], with MGT staining. A histochemical re-
action for NADH shows the presence of moth-eaten fibers [H]. The predominance of type I fibers was
seen with an ATPase reaction (pH 4.3) [I]. (A, D, E, F, H, I: case 3; B, C, G: case 8). Bar = 50µm.
Fig 2. Immunofluorescence changes in mus-
cle fibers with nemaline myopathy: [A/B]
focal faults of nebulin, [C] absence of neb-
ulin, [D] normal myosin (mosaic pattern),
[E] strongly positive desmin, and [F] nor-
mal desmin. Bar = 50 µm.
Arq Neuropsiquiatr 2009;67(3-B)
Youssef et al.
distribution of rods was mixed (subsarcolemmal and in-
termyofibrilar) in all cases (Fig 1).
Immunohistochemical analysis showed abnormal re-
sults for nebulin, myosin and desmin muscle proteins in
all cases (Table). Nebulin showed heterogeneous expres-
sion in four cases and was absent in the other four cases
(Fig 2 / Table). We never observed homogeneous expres-
sion of nebulin. Desmin expression was homogeneous in
four cases, strongly positive in three cases, and absent in
one case (Fig 2 / Table). Fast myosin expression showed
a mosaic pattern in six cases and was absent in two cases
(Fig 2 / Table). The relationship between the clinical form
of NM and nebulin, desmin and fast myosin expression on
muscle biopsy is shown in Table.
NM is a rare form of congenital myopathy. The small
number of cases found in our study is illustrative of the
Table. Relationship between the clinical form and characteristics of NM patients.
1Number of patients
Obstetrical history / Gestational complications
Decreased fetal movements
Predominance of type I fibers
Focal increase in acid phosphatase in fibers
NM: nemaline myopathy, CK: creatine kinase, LDH: lactate dehydrogenase.
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Youssef et al.
low incidence of NM of 1 case per 500000 live births25.
We identified a higher number of female patients than
male patients (six female and two male), although the lit-
erature does not suggest any gender predominance.
In the series described by Gommans et al. and Wall-
gren-Pettersson, there was a higher incidence of the typ-
ical form of NM3,6. Although this is a rare disease, the typ-
ical form has the highest incidence, followed by the inter-
mediate congenital form and the severe congenital form.
In this study, all of the patients showed mild to severe
hypotonia from birth onward, confirming reports of oth-
er series of patients with NM1-3, 6,7,13.
With regard to muscle enzymes, CK was normal or
slightly elevated (up to five times the normal value), and
there was no muscle necrosis or muscle regeneration pat-
tern detected in biopsies of NM patients. This has also
been observed in most other cases5,6.
The EMG examination typically shows diverse fea-
tures in NM patients monitored throughout the course
of the disease6,29. Before the age of 3 years, the EMG usu-
ally shows mild abnormalities and a myopathic pattern
(the motor unit potential (MUP) has a short duration, low
amplitude, increased recruitment and short polyphasic
potentials) can be found in a few patients. Between the
ages of 3 and 10 years, there is an increase in the inci-
dence of abnormalities, although there are many normal
MUPs, myopathic MUPs are predominant. After the age of
11 years, there are few normal MUPs and a neurogenic pat-
tern (MUPs with long duration, high amplitude, decreased
recruitment and long polyphasic potentials) can occur. In
adults with NM, these features are primarily seen in dis-
tal muscles such as the tibialis anterior. Spontaneous mus-
cle activity such as fibrillation and positive waves are rare
in NM. These abnormalities suggest a progressive form of
primary myopathy probably due to motor unit remodel-
ing leading to secondary neuropathy changes. In our cas-
es, the patients with normal EMG were under 2 years old,
consistent with the literature. The patients with myopath-
ic and neurogenic patterns, however, were between the
ages of 2 months and 3.5 years, suggesting premature EMG
abnormalities in our group6,24.
Nemalin bodies or rods are a sign of NM, and the best
technique for detecting them is the MGT staining of biop-
sy sections1,2,5,11. With other staining or histochemical tech-
niques, such as ATPase staining, the results are often not
distinct or can show a mild negative image. Nemaline bod-
ies are distributed randomly along the muscle fiber, but
have a tendency to be attached below the sarcolemma
(subsarcolemmal location), or appear as difused spots in
the interior of the fiber (intermyofibrilar location). Mixed
patterns can also occur. Intranuclear rods have been de-
scribed in severe neonatal and adult forms26,30. Rods were
identified in less than 50% of muscle fibers in six of our
patients and more than 50% in two of our patients via
MGT staining. All cases showed a mixed location pattern,
regardless of the NM clinical form.
The predominance of type I fibers is a common feature
in NM, and some patients can have exclusively type I fibers
or poor differentiation between fibers5,12-14,. Infrequently,
there is a focal increase in acid phosphatase activity in fi-
bers, which is associated with cases with a fast evolution.
This suggests a degenerative process, inducing lysosom-
al enzyme activation and celular destruction13,14. Our cas-
es showed a predominance of type I fibers and increased
acid phosphatase activity, regardless the NM clinical form.
The analysis of nebulin protein expression using immu-
nohistochemical began in the last decade and there are
descriptions of normal and abnormal expression patterns
in patients with NM, mainly for the typical form15-17. Neb-
ulin acts as a molecular ruler for thin filaments and is im-
portant for the assembly and integration of Z discs with
the sarcomere15,17. In our study, the typical form of NM was
associated with abnormal nebulin expression, as were the
severe and intermediate congenital forms, which is similar
to reports in other studies15-17. For desmin protein expres-
sion, the presence of areas of accumulation in muscle fi-
bers is a pathological finding and may represent immatu-
rity or a muscle fiber regeneration process. Van der Ven
described strongly positive desmin expression in cases of
severe congenital NM, while in mild cases, there was not a
distinct expression pattern18. In our study, both the severe
congenital form and the typical and intermediate congen-
ital forms showed strongly positive desmin expression in
muscle fibers. Fast myosin expression was absent in two
patients, while positive expression with a mosaic pattern
was observed in the majority of patients.
Six NM-related genes, all encoding components of the
thin filament of the sarcomere, have been described. Stud-
ies of several families with severe, intermediate and typi-
cal NM forms have identified mutations in α-tropomyosin
(TPM3) and beta-tropomyosin19-21. A recent study de-
scribed an Australian familly with a mutation in the TPM3
gene22. Several mutations were identified in the nebulin
(NEB) gene due to small deletions or insertions22,23. Muta-
tions in the α-actin gene (ACTA1) may be related to con-
genital fiber type disproportion24,25, and have been report-
ed to be exclusively associated with intranuclear rods26.
Troponin T gene (TNNT1) mutations were described in an
isolated group of old order Amish NM patients4, 24. A sixth
gene, CFL2, encoding the actin-binding protein muscle co-
filin-2, was reported in two siblings with congenital myo-
pathy27. Our patients could have had any of these genetic
alterations. Our patients had abnormal nebulin expression
by immunohistochemistry suggesting mutations in nebu-
lin. Mutations in CFL2, ACTA1 and TNNT1 are less probable
to occur in our patients because we had only one case of
Arq Neuropsiquiatr 2009;67(3-B) Download full-text
Youssef et al.
the congenital form of NM, and there were no cases with
intranuclear rods or Amish patients. Unfortunately, mu-
tations in many of these genes do not predict the severi-
ty or prognosis of the disease and do not determine pat-
terns of inheritance31.
In the present study, we focused on protein expression
analysis in patients with NM and concluded that regard-
less of the NM clinical form, nebulin may have heteroge-
neous or absent protein expression, and fast myosin pro-
tein expression may be positive with a mosaic pattern in
the majority of patients. In addition, the desmin protein,
which is related to the maturity level of the muscle, may
show normal or abnormal expression in NM patients.
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