2014, Volume 30, Number 2, Page(s) 111-117
Histopathological and Genetic Features of Patients with Limb Girdle Muscular Dystrophy Type 2C
Gülden DİNİZ1, Filiz HAZAN2, Hülya TOSUN YILDIRIM3, Aycan ÜNALP4, Muzaffer POLAT5, Gül SERDAROĞLU6, Orkide GÜZEL4, Özlem BAĞ4, Yaprak SEÇİL7, Figen ÖZGÖNÜL1, Sabiha TÜRE7, Galip AKHAN7, Ajlan TÜKÜN8
1Tepecik Education and Research Hospital, Neuromuscular Disease Center, İZMİR, TURKEY
2Department of Medical Genetics, Dr. Behçet Uz Children's Hospital, İZMİR, TURKEY
3Department of Pathology, Dr. Behçet Uz Children's Hospital, İZMİR, TURKEY
4Department of Pediatric Neurology, Dr. Behçet Uz Children's Hospital, İZMİR, TURKEY
5Department of Pediatric Neurology,Celal Bayar University, Faculty of Medicine, Manİsa, Turkey
6Ege University, Faculty of Medicine, İZMİR, TURKEY
7Department of Neurology, Katip Çelebi University, Faculty of Medicine, İZMİR, TURKEY
8Department of Medical Genetics, Düzen Laboratory, ANKARA, TURKEY
Keywords: Dystrophin, Gamma sarcoglycan, Genetic testing, Muscular dystrophy, Limb-Girdle
In this study, it was aimed to describe the clinical,
histopathological and genetic features of 20 patients with gamma
sarcoglycanopathy confirmed by muscle biopsies and genetic analysis.
Material and Method: We retrospectively reviewed 20 patients from
whom muscle biopsy specimens were obtained between 2007 and
2012. All patients were clinically diagnosed as muscular dystrophy
and biopsy materials were collected from five different centers of
neurological disorders. All DNAs were extracted from muscle tissues
or blood samples of patients and genetic tests (mutation analyses for
gamma sarcoglycan gene and deletion-duplication analyses for all 4
sarcoglycan genes) were performed.
Results: The mean age of the patients was 7.6 years (2 -21 years).
Only one case (5%) was older than 14 years. The mean CPK level was
10311 U/L (1311 35000 U∕L). There were 4 siblings in these series.
Expression defects of gamma sarcoglycan staining were determined in
(15 males, and 5 females) all patients with muscle biopsy specimens.
But only in 9 of them, disease-causing defects could be determined
with genetic analyses.
Conclusion: The present study has demonstrated that both
examination of muscle biopsy specimens and DNA analysis remain
important methods in the differential diagnosis of muscular
dystrophies. Because dystrophinopathies and sarcoglycanopathies
have similar clinical manifestation.
Gamma sarcoglycan (γ-SGC) is one of the four sarcoglycans
(SGCs) found at the cell membrane of skeletal muscle. The
SGCs form a subcomplex closely linked to the dystrophinassociated
glycoprotein complex (DAG). Proper presence
of SGCs is essential for membrane integrity during muscle
contraction. Limb girdle muscular dystrophy type 2C
(LGMD-2C) is an autosomal recessive muscle-wasting
disorder caused by genetic defects in the sarcoglycan
gamma (SGCG) gene. It is also known as the childhood
severe muscular dystrophy and clinically resembles the
dystrophinopathies which are the most common muscular
No definitive treatments for the LGMD-2C and the other
muscular dystrophies exist. Management to prolong
survival and improve quality of life includes physical
therapy, and stretching exercises to promote mobility
and prevent contractures, weight control to avoid obesity,
surgery for orthopedic complications, use of mechanical
and respiratory aids to help ambulation, mobility and
respiration. Monitoring cardiomyopathy for cardiac
involvement and emotional support are also required5-9.
Differential diagnosis of LGMD-2C is made in consideration
of Duchenne and Becker muscular dystrophies
(DMD/BMD) and it is impossible to differentiate between
these conditions solely on clinical grounds. Therefore immunohistochemical
staining of muscle biopsy specimens
and molecular genetic analysis are mandatory for correct
diagnosis (10-12). In this study, we aimed to determine
the spectrum of genetic defects in immunohistochemically
proven cases of LGMD-2C, to correlate the findings with
clinical phenotypes and to display the regional differences
as for the clinical, histopathological, and genetic characteristics
of gamma sarcoglycanopathies.
Histopatological examinations of muscle biopsies were
performed at Pathology Laboratory of Izmir Dr. Behçet
Uz Childrens Hospital. Genetic analyses were performed
at Ankara Düzen Laboratory from January 2007 through
December 2012. Twenty patients with defective gamma
sarcoglycan expressions found on the muscle biopsy
specimens, and clinically diagnosed as muscular dystrophy
were included in this study. Immunohistochemical analysis
(IHC) was repeated to confirm the diagnosis. Individual
patient database was reviewed in all cases, and clinical
information of patients was recorded including age, gender,
detailed family history and consanguinity. Neurological
examination and laboratory findings were also evaluated.
Laboratory evaluation included serum creatine kinase (CK),
serum aspartate aminotransferase (AST) analyses, and
nerve conduction and electromyographic (EMG) studies.
All muscle biopsies were obtained from the gastrocnemius
Samples were frozen in isopentane cooled in liquid nitrogen
and 8- to 12- micron sections were cut using the cryostat.
Slides were stained with hematoxylin-eosin (H&E), as
well as with several histochemical and enzymatic stains.
Cryosections were immunostained for dystrophin using
a polyclonal antibody (Neomarkers) with a monoclonal
spectrin antibody (Novocastra) as a control. SGCs were
detected with anti alpha (α-), beta (β-), delta (δ-) and γ-
SGC antibodies (Novocastra).
Genomic DNAs were extracted from the remnant muscle
tissues or blood samples using available DNA extraction
kits (QiaGen, US) following the manufacturers standard
protocol. The exon regions and flanking short intronic
sequences of the SGCG gene were amplified using the
polymerase chain reaction (PCR), followed by direct
sequencing of the PCR products (ABI, US). In addition, the
multiplex ligation-dependent probe amplification (MLPA)
technique was used for deletion and duplication analysis for
all 4 SGCs.
Frequencies and descriptive analyses were performed using
the statistical software SPSS 9.05 for Windows.
Twenty patients with severe muscle disease were evaluated
respectively. All of them had been diagnosed as muscular
dystrophy on the basis of muscle biopsy findings. Severe
alterations of myofiber size and shape, splitting, increase
in the number of internal nuclei, fiber type disproportions;
necrosis, myophagocytosis, regeneration and fibrosis were
simply classified as muscular dystrophy (Figure 1
Click Here to Zoom
|Figure 1: Note the regenerated muscle fibers which are specific for
muscular dystrophies (H&E, x100).
The mean age of the patients was 7.6 years (2 to 21 years).
There were 4 siblings (n=8) in these series. Expression
defects of gamma sarcoglycan staining were determined in
(15 male and 5 female) all patients with available muscle
biopsy specimens. However, disease-causing defects could
be determined with genetic analyses in only 9 of them. The
mean age of the patients was 7.6 years (± 4.11), ranging from
2 to 21 years. Only one case (5%) was older than 14 years.
The detailed clinical characteristics of the patients were
presented in Table I. All patients presented some degree
of muscle weakness. All of them had high creatine kinase
(CPK) levels. The mean CPK level was 10311 U/L (1311
35000 U/L). Ten patients (50%) had similarly affected family members. The consanguinity rate was 45% (n=9). Physical
examination at the time of diagnosis revealed weakness
in proximal limb muscles. Needle electromyogram was
performed and revealed myopathy in all patients. All
patients were ambulatory at the time of diagnosis.
The final diagnosis was made on the basis of muscle biopsy
findings. All twenty cases showed staining defects for
gamma sarcoglycan with the presence of staining for other
sarcoglycans and dystrophin (Figure 2A-D). Similarly there
were no defects in the dystrophin genes. Although there
were defective expressions of gamma sarcoglycan protein
in all biopsy specimens, the disease-causing genetic defects
could be determined in only nine of them. Most cases had
silent homozygous or heterozygous mutations. The detailed
genetic defects of the patients are presented in Table II.
Click Here to Zoom
|Figure 2: a) Nearly normal expressions
of α-SGC, b) β-SGC, c) δ-SGC and
d) Defective expression of sarcolemmal
γ-SGC (DAB, x100).
The human SGCG gene is located on chromosome 13q12. It
consists of 8 exons. The sequence of SGCG is composed of
291 amino acids. Three portions of the gamma sarcoglycan
extracellular domain display possible critical function, two
for the assembly with either beta or alpha sarcoglycan, and
the putative EGF-like domain. Hitherto forty mutations
have been described in the gamma sarcoglycan gene2
The homozygous del525T mutation generates a truncated
gamma sarcoglycan protein without EGF-like domain, which is able to assemble with the other sarcoglycans1,2,13-15
. This mutation is commonly found in North
Africa. The C283Y mutation in the cystein-rich domain
could be functionally relevant, because this cysteine is
crucial for the EGF-like domain. The C283Y mutation can
cause severe LGMD and it is the most common mutation in
the Gypsy ethnic groups of the Europe1,2,15-17
. In the
present study, we have determined homozygous del525T
mutation in a sibling, but we could not find a C283Y
Immunohistochemical analysis of the sarcolemmal proteins
such as dystrophin, SGCs, merosin, and dysferlin is
an important part of the diagnostic evaluation of muscle
biopsies in patients with muscular dystrophy. Reduced or
absent sarcolemmal expression of one of the 4 SGCs can
be found in patients with any LGMDs and also in patients
with dystrophinopathies. It has been previously suggested
that different patterns of SGC expression could predict the
primary genetic defect, and that genetic analysis could be
directed by these patterns1,12. However Klinge et al.10
reported that residual SGC expression could be highly variable
and an accurate prediction of the genotype could not
be achieved. Therefore they recommended using antibodies
against all four SGCs for immunoanalysis of skeletal muscle
sections. Similarly, a concomitant reduction of dystrophin
and any one of SGCs may have a crucial importance in the
differential diagnosis of dystrophinopathies for sarcoglycan
deficient LGMD1-5. For this reason, it is not easy to
decide whether the disease is a dystrophinopathy with defective
expressions of SGCs or a LGMD with defective expression
of dystrophin. Since in the cases of this series, the
sarcolemmal dystrophin staining and dystrophin gene were
not abnormal, Duchenne Muscular Dystrophy (DMD) or
Becker Muscular Dystrophy (BMD) were not considered
for differential diagnosis.
Patients with any LGMD may be clinically indistinguishable
from those with primary dystrophinopathies. Probably,
the diagnosis of LGMD has been underestimated
and a number of male patients were diagnosed as DMD or
BMD1,3,7. If a definitive diagnosis can be made based
on appropriate immunohistochemical examinations and
molecular analysis performed in those patients, a normal
staining pattern of dystrophin and an autosomal recessive
mode of inheritance can be determined. On the contrary,
patients with dystrophinopathy may show variable findings
from normal to regional absence or mosaic pattern of sarcolemmal
staining with anti-SGCs antibodies which signify
different presentation of abnormal organization of the cellmembrane
associated dystrophin glycoprotein complex.
Therefore careful examination of immunohistochemical
staining with genetic study is necessary to make an accurate
In summary, this study adds different mutations to the
growing list of defects that can be associated with LGMD-
2C and further emphasizes the importance of systematic
analysis of all related genes, instead of analyzing only the
primarily deficient SGCs gene. In this study, we have also
highlighted the patterns of genetic complexity associated
with LGMD-2C encountered during the process of
differential diagnosis of muscular dystrophies18.
CONFLICT of INTEREST
The authors had received financial support from Izmir
Katip Çelebi University.
1) Dubowitz V, Sewry CA, Oldfors A. Muscle Biopsy: A practical
approach. Philadelphia: Saunders; 2013.276-302.
2) Sandonà D, Betto R. Sarcoglycanopathies: Molecular pathogenesis
and therapeutic prospects. Expert Rev Mol Med. 2009;11:e28. [ PubMed ]
3) Ben Jelloun-Dellagi S, Chaffey P, Hentati F, Ben Hamida
C, Tome F, Colin H, Dellagi K, Kaplan JC, Fardeau M, Ben
Hamida M. Presence of normal dystrophin in Tunisian severe
childhood autosomal recessive muscular dystrophy. Neurology.
1990;40:1903. [ PubMed ]
4) Pogue R, Anderson LV, Pyle A, Sewry C, Pollitt C, Johnson MA,
Davison K, Moss JA, Mercuri E, Muntoni F, Bushby KM. Strategy
for mutation analysis in the autosomal recessive limb-girdle
muscular dystrophies. Neuromuscul Disord. 2001;11:80-7. [ PubMed ]
5) Moreira ES, Vainzof M, Suzuki OT, Pavanello RC, Zatz M, Passos-
Bueno MR. Genotype-phenotype correlations in 35 Brazilian
families with sarcoglycanopathies including the description of
three novel mutations. J Med Genet. 2003;40:E12. [ PubMed ]
6) Gulati S, Leekha S, Sharma MC, Kalra V. Gamma-sarcoglycanopathy.
Indian Pediatr. 2003;40:1077-81. [ PubMed ]
7) Azibi K, Bachner L, Beckmann J S, Matsumura K, Hamouda E,
Chaouch M, Chaouch A, Ait-Ouarab R, Vignal A, Weissenbach
J. Severe childhood autosomal recessive muscular dystrophy with
the deficiency of the 50 kDa dystrophin-associated glycoprotein
maps to chromosome 13q12. Hum Molec Genet. 1993;2:1423-28. [ PubMed ]
8) El Kerch F, Sefiani A, Azibi K, Boutaleb N, Yahyaoui M, Bentahila
A, Vinet MC, Leturcq F, Bachner L, Beckmann J, et al. Linkage
analysis of families with severe childhood autosomal recessive
muscular dystrophy in Morocco indicates genetic homogeneity
of the disease in North Africa. J Med Genet. 1994;31: 342-3. [ PubMed ]
9) Lasa A, Piccolo F, de Diego C, Jeanpierre M, Colomer J, Rodríguez
MJ, Urtizberea JA, Baiget M, Kaplan J, Gallano P. Severe limb
girdle muscular dystrophy in Spanish gypsies: Further evidence
for a founder mutation in the gamma-sarcoglycan gene. Eur J
Hum Genet. 1998;6:396-9. [ PubMed ]
10) Klinge L, Dekomien G, Aboumousa A, Charlton R, Epplen JT,
Barresi R, Bushby K, Straub V. Sarcoglycanopathies: Can muscle
immunoanalysis predict the genotype? Neuromuscul Disord.
2008;18:934-41. [ PubMed ]
11) Trabelsi M, Kavian N, Daoud F, Commere V, Deburgrave N,
Beugnet C, Llense S, Barbot JC, Vasson A, Kaplan JC, Leturcq F,
Chelly J. Revised spectrum of mutations in sarcoglycanopathies.
Eur J Hum Genet. 2008;16:793-803. [ PubMed ]
12) Ferreira AF, Carvalho MS, Resende MB, Wakamatsu A,
Reed UC, Marie SK. Phenotypic and immunohistochemical
characterization of sarcoglycanopathies. Clinics (Sao Paulo).
2011;66:1713-19. [ PubMed ]
13) Jung D, Leturcq F, Sunada Y, Duclos F, Tome FMS, Moomaw C,
Merlini L, Azibi K, Chaouch M, Slaughter C, Fardeau M, Kaplan
JC, Campbell KP. Absence of gamma-sarcoglycan (35 DAG) in
autosomal recessive muscular dystrophy linked to chromosome
13q12. FEBS Lett. 1996:381: 15-20.
14) Kefi M, Amouri R, Driss A, Ben Hamida C, Ben Hamida M,
Kunkel LM, Hentati F. Phenotype and sarcoglycan expression in
Tunisian LGMD 2C patients sharing the same del521-T mutation.
Neuromuscul Disord. 2003;13:779-87.
15) Navarro C, Teijeira S. Neuromuscular disorders in the Gypsy
ethnic group: A short review. Acta Myol. 2003; 22: 11-14.
16) McNally EM, Duggan D, Gorospe J R, Bonnemann C G, Fanin
M, Pegoraro E, Lidov HG, Noguchi S, Ozawa E, Finkel RS, Cruse
RP, Angelini C, Kunkel LM, Hoffman EP. Mutations that disrupt
the carboxyl-terminus of gamma-sarcoglycan cause muscular
dystrophy. Hum Molec Genet. 1996;5:1841-7.
17) Merlini L, Kaplan JC, Navarro C, Barois A, Bonneau D, Brasa
J, Echenne B, Gallano P, Jarre L, Jeanpierre M, Kalaydjieva L,
Leturcq F, Levi-Gomes A, Toutain A, Tournev I, Urtizberea A,
Vallat JM, Voit T, Warter JM. Homogeneous phenotype of the
gypsy limb-girdle MD with the gamma-sarcoglycan C283Y
mutation. Neurology. 2000;54:1075-9.
18) Dinçer P, Akçören Z, Demir E, Richard I, Sancak O, Kale G, Ozme
S, Karaduman A, Tan E, Urtizberea JA, Beckmann JS, Topaloğlu
H. A cross section of autosomal recessive limb-girdle muscular
dystrophies in 38 families. J Med Genet. 2000;37:361-7.