Summary

Objective: 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.

Introduction

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 dystrophies[1-4].

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 required[5-9].

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.

Methods

Histopatological examinations of muscle biopsies were performed at Pathology Laboratory of Izmir Dr. Behçet Uz Children’s 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 muscle.

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 manufacturer’s 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.

Results

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).

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.

Table I: Clinical and genetic features of patients

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.

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).

Table II: Nucleotide and amino acid sequences of SGCG gene

Discussion

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 gene[2]. The homozygous del525T mutation generates a truncated gamma sarcoglycan protein without EGF-like domain, which is able to assemble with the other sarcoglycans[1,2,13-15]. 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 Europe[1,2,15-17]. In the present study, we have determined homozygous del525T mutation in a sibling, but we could not find a C283Y mutation.

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 patterns[1,12]. 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 LGMD[1-5]. 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 BMD[1,3,7]. 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 diagnosis[1,2].

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 dystrophies[18].

CONFLICT of INTEREST
The authors had received financial support from Izmir Katip Çelebi University.

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