The Histopathologic Examination of a Second Muscle Biopsy Specimen at a Later Date may Sometimes be the Best Approach to Make a Differential Diagnosis in Neuromuscular Disorders
Gulden DINIZ1, Berk OZYILMAZ2, Sarenur GOKBEN3
1İzmir Demokrasi University, School of Medicine, Department of Pathology, IZMIR, TURKEY
2University of Health Sciences, İzmir Tepecik Education and Research Hospital, Medical Genetics Lab., IZMIR, TURKEY
3Ege University, School of Medicine, Department of Pediatric Neurology, IZMIR, TURKEY
Neuromuscular disorders still keep their mystery 1.
Considering that cases with very mild symptoms cannot be
diagnosed at all, it is almost impossible to know the true
prevalence of these diseases 2. Relatively little information
about the exact prevalence of neuromuscular disorders
(NMDs) has been published 1-4. It has been reported
that NMDs affect approximately one in 3500 children
worldwide and X-linked dystrophinopathies have the
highest incidence among them 4. Knowledge of NMDs
has expanded dramatically during the last four decades
thanks to advances in modern pathological techniques and
genetic tests. Currently, the dystrophinopathies and most
cases of limb-girdle dystrophies (LGMDs) can be diagnosed
with immunohistochemical analysis of muscle tissues 4-7.
It must be kept in mind that diagnoses may be suggested
by the histopathological evaluation, but definitive diagnosis
mostly relies on genetic analyses 5-7.
It can be thought that the evaluation by molecular
techniques of comprehensive panels, each aimed at
identifying genetic changes in a separate disease group,
reduces the importance of pathological examination of
muscle tissue 6. However, the evaluation of muscle biopsy
specimens is an irreplaceable approach in some cases. For
example, dystrophinopathy, the most common muscular
disease, has two clinically and significantly different
entities: Duchenne (DMD) and Becker muscular dystrophy
(BMD). Among these two diseases that start in childhood,
patients with DMD die in the second decade of their lives,
and patients with BMD can reach their 50s thanks to close
surveillance. Interestingly, genetic changes are similar
in both diseases and cannot be distinguished by genetic
analyses 4. The only way to differentiate the two diseases
is to show the complete loss of dystrophin as in DMD or
the presence of partially functional dystrophin protein as in
BMD. Therefore, demonstration of sarcolemmal dystrophin
in muscle biopsy tissue specimens has vital diagnostic
importance 5. Similarly, the immunohistochemical
examination of muscle biopsy specimens with commercially
available primary antibodies is helpful in the diagnosis of
many muscular dystrophies. In addition, staining of muscle
biopsy specimens with combined succinate dehydrogenasecytochrome oxidase (SDH-COX) enzyme stain is the
most reliable way to diagnose mitochondrial myopathy in
mitochondrial diseases with muscle involvement. Oil red
O and PAS stains are also helpful for differential diagnosis
of muscle involvement in metabolic diseases. In this way,
unnecessary treatments can be avoided in many myopathies
considered in the differential diagnosis 5-7.
Histopathologic evaluation of muscle biopsy specimens
is an important step in the diagnosis of a neuromuscular
disease. An experienced pathologist can provide very
useful clues to the clinician for differential diagnosis.
Presence of inflammatory cells or regenerated fibers, group
atrophy, ragged red fiber, core or targets, increase in the
number of central nuclei, accumulation of glycogen or
lipid and fibrosis-like pathologies can be determined in
neuromuscular disease. These findings discriminate against
the disease group and determine the most useful genetic
analysis that may be preferred for further examination.
Especially in children older than 6-8 weeks, the presence
of muscle fibers showing neonatal myosin expression in
immunohistochemical analysis provides further evidence
for a muscle disease 5-7.
Single muscle biopsy is almost always sufficient. For
additional diagnostic approaches, the biopsy material
stored at -80 oC should be re-evaluated by an experienced
muscle pathologist with new dyes that have not been applied
before. Repeat biopsy examination may be required only
in newborn infants. In many muscle diseases, including
DMD, recurrent biopsies only show a progression to an
end-stage muscle disease and eventually the muscle tissue
is replaced by fibroadipose tissue. There are few muscle
diseases in which histopathologic examination of repeat
biopsy specimens has diagnostic value. For example, in
centronuclear myopathy, the nuclei in the muscle fibers
are located peripherally at birth but migrate to the center
in the following years, which is considered as a kind of
dedifferentiation. Diagnosis cannot be made based on the
histopathologic examination of the first biopsy specimens
obtained in early childhood, and the diagnosis is usually
established based on the examination of biopsy specimens
harvested around the age of 4 1-3.
A muscle biopsy specimen of a 10-month-old boy was
evaluated histopathologically nearly five years ago. He
was a hypotonic infant and had respiratory distress.
There was no consanguinity between his parents. In the
histopathologic examination of the first biopsy material,
the presence of an increased number of central nuclei
and myofibers of various sizes were detected. The patient
was evaluated as a case of congenital myopathy such as
centronuclear myopathy, myofiber-type disproportion and
so on. In the first biopsy, there were mild differences in the
size and shape of fibers. Numerous internal nuclei were not
observed (Figure 1A-C). A repeat biopsy was suggested
at a later date. Two years later, a new biopsy material was
available. Interestingly, in the histopathologic examination
of the second biopsy specimens, greater differences were
seen in the sizes of fibers. Small fibers were of type1 and
all fibers always had internal nuclei. Muscle injury had
deteriorated to muscular dystrophy (Figure 2A-C). As the centronuclear myopathy must not have dystrophic
features, and exaggerated differences in fiber sizes as seen
in this case, the slides were consulted with an expert muscle
pathologist, Professor Caroline Sewry, Ph.D., FRCPath,
about the biopsy results who suggested a genetic evaluation.
She wrote that central nuclei and small slow fibers seemed
to be the main features, so a panel of congenital myopathy
including MTM1, Titin (TTN), and RYR1 genes must be
evaluated. Nearly a year later, we genetically evaluated these
three genes and determined a homozygous MTM1 gene
mutation (c.731C>T p.R241C). This variant was previously
reported as pathogenic. Finally, the patient was diagnosed
as a case of centronuclear myopathy caused by the mutation
of the MTM1 gene on Xq28 6.
Click Here to Zoom
|Figure 1: The first biopsy material. A) There are mild differences of myofiber size and a mild increase in internal nuclei (H&E; x100).
B) The larger myofibers have type 2 features by anti-fast myosin antibody (IHC; x 100). C) Normal sarcolemmal dystrophin expression
(IHC; x 100).
Click Here to Zoom
|Figure 2: The second biopsy material. A) There are severe differences of myofiber size and most myofibers have internal nuclei (H&E;
x200). B) The huge myofibers have type 2 features by anti- fast myosin antibody (IHC; x200). C) Normal sarcolemmal dystrophin
expression and very prominent internal nuclei (IHC; x200).
In conclusion, unlike many other diseases, the histopathologic
examination does not form the basis of the diagnosis
of neuromuscular diseases. As in many parts of the world, a limited number of centers and a scarce number of trained
pathologists are performing special muscle and nerve biopsy
examinations in our country. Nevertheless, as observed
in the present case, histopathologic examination, and more
importantly, repeat biopsy is invaluable in the differential
diagnosis. For this reason, it is very important to be able
to perform the histopathologic examination of muscle and
nerve biopsy specimens at least in some large centers and to
provide the relevant training to the pathologists interested
in this subject.
We thank Prof. Caroline Sewry Ph.D. FRCPath for her
contribution to making the diagnosis of the patient.
This article was edited in English by Gurkan Kazanci,
M.D., Ph.D., who is a professional medical translator and
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