Concomitant Alpha- and Gamma-Sarcoglycan Deficiencies in a Turkish Boy with a Novel Deletion in the Alpha-Sarcoglycan Gene

Abstract

Limb-girdle muscular dystrophy type 2D (LGMD-2D) is caused by autosomal recessive defects in the alpha-sarcoglycan gene located on chromosome 17q21. In this study, we present a child with alpha-sarcoglycanopathy and describe a novel deletion in the alpha-sarcoglycan gene. A 5-year-old boy had a very high serum creatinine phosphokinase level, which was determined incidentally, and a negative molecular test for the dystrophin gene. Muscle biopsy showed dystrophic features. Immunohistochemistry showed that there was diminished expression of alpha- and gamma-sarcoglycans. DNA analysis revealed a novel 7 bp homozygous deletion in exon 3 of the alpha-sarcoglycan gene. His parents were consanguineous heterozygous carriers of the same deletion. We believe this is the first confirmed case of primary alpha-sarcoglycanopathy with a novel deletion in Turkey. In addition, this study demonstrated that both muscle biopsy and DNA analysis remain important methods for the differential diagnosis of muscular dystrophies because dystrophinopathies and sarcoglycanopathies are so similar.

1. Introduction

Limb girdle muscular dystrophy type 2D (LGMD-2D) is an autosomal recessive muscular disease caused by genetic defects in sarcolemmal alpha sarcoglycan (α-SGC) glycoprotein. Alpha-SGC or adhalin, one of the four sarcoglycans (SGCs), is essential for membrane integrity during muscle contraction and provides a scaffold for important signaling molecules [1–3]. Alpha-SGC is encoded by the sarcoglycan alpha gene (SGCA) located on chromosome 17q21 [1, 4]. LGMD-2D predominantly affects proximal muscles around the scapular and the pelvic girdles. LGMD-2D has a very heterogeneous phenotype. The age of onset, rate of progression, and the severity of disease can vary between and also within affected families. The most clinically severe course is generally observed when the sarcolemmal α-SGC is totally absent whereas milder phenotypes are observed when residual proteins are present [1–4]. Interestingly, a mutation in any SGC gene can lead to a reduction or absence of the other SGCs [4–7]. It was previously reported that the SGCA gene must be evaluated first if there is a concomitant absence of α-SGC and gamma- (γ-) SGC [4].

The differential diagnosis for LGMD-2D includes Duchenne and Becker muscular dystrophies (DMD/BMD) and it is impossible to reach a diagnosis on clinical grounds alone. Therefore, immunohistochemical staining of a muscle biopsy and molecular genetic analysis are mandatory for the correct diagnosis [3, 5, 8, 9]. In this report, the patient's genotype was a previously unknown 7 bp deletion in exon 3. This finding adds to the growing spectrum of mutations in the alpha-sarcoglycan gene. Finally, we also discuss important considerations in the differential diagnosis of the muscular dystrophies.

2. Case Report

A 5-year-old boy had second degree consanguineous parents from Turkey without an ancestral history of neuromuscular disorders. There were no complications during pregnancy, and antenatal signs of muscular disorders such as polyhydramnios and reduced fetal movements were not noted. Cognitive and motor development was normal. At the time of presentation, his previously undetected mild muscle weakness was predominantly proximal. Deep tendon reflexes were present and he had no contractures. He was walking normally but he had mild difficulty when climbing stairs and running. Pulmonary function tests were normal. His creatinine phosphokinase (CPK) levels were between 9000 and 15000 units per liter (normal < 250 U/L), and there were myopathic changes on electromyography. Because of the very high CPK level, muscular dystrophy was suspected and, after informed consent, samples were obtained for histopathology, immunohistochemistry, and molecular genetics testing.

A muscle biopsy specimen from the left gastrocnemius muscle of the patient was frozen in isopentane that was precooled to −160°C in liquid nitrogen. Cryosections were immunostained for dystrophin using a polyclonal antibody (Neomarkers), with a monoclonal spectrin antibody (Novocastra) as a control. A neonatal myosin heavy chain (Neonatal myosin, Novocastra) antibody was used for the identification of pathological immature myofibers. SGCs were detected with anti-α-, -β-, -δ-, and -γ-SGC antibodies (Novocastra).

Peripheral blood specimens were collected from the proband and parents. Genomic DNA was extracted from whole blood using a commercial DNA extraction kit (QiaGen, USA) following the standard manufacturer's protocol. The concentration of sample DNA was determined by a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). The exon regions and flanking short intronic sequences of the SGCA gene were amplified using polymerase chain reaction (PCR), followed by direct sequencing of the PCR products (ABI, US) (NCBI Reference Sequence: NG_008889.1). Hitherto reported genetic abnormalities in LGMD-2D are listed in Table 1.

Table 1.

Nucleotide and amino acid sequences of α-SGC gene.

1 1

ATG Met

GCT Ala

GAG Glu

ACA Thr

CTC Leu

TTC Phe

TGG Trp

ACT Thr

CCT Pro

CTC Leu

CTC Leu

GTG Val

GTT Val

CTC Leu

CTG Leu

46 16

GCA Ala

GGG Gly

CTG Leu

GGG Gly

GAC Asp

ACC Thr

GAG Glu

GCC Ala

CAG Gln

CAG Gln

ACC Thr

ACG Thr

CTA Leu

CAC His

CCA  Pro#

91 31

CTT  Leu#

GTG Val

GGC Gly

CGT  Arg#

GTC Val

TTT Phe

GTG Val

CAC His

ACC Thr

TTG Leu

GAC Asp

CAT His

GAG Glu

ACG Thr

TTT Phe

136 46

CTG Leu

AGC Ser

CTT Leu

CCT Pro

GAG Glu

CAT His

GTC Val

GCT  Ala#

GTC Val

CCA Pro

CCC Pro

GCT Ala

GTC Val

CAC His

ATC Ile

181 61

ACC Thr

TAC  Tyr#

CAC His

GCC Ala

CAC His

CTC Leu

CAG Gln

GGA  Gly#

CAC His

CCA Pro

GAC Asp

CTG Leu

CCC Pro

CGG  Arg#

TGG Trp

226 76

CTC Leu

CGC  Arg#

TAC  Tyr##

ACC  Thr##

CAG  Gln##

CGC  Arg#

AGC Ser

CCC Pro

CAC His

CAC His

CCT Pro

GGC Gly

TTC Phe

CTC  Leu#

TAC  Tyr#

271 91

GGC  Gly#

TCT Ser

GCC  Ala#

ACC Thr

CCA Pro

GAA Glu

GAT  Asp#

CGT  Arg#

GGG Gly

CTC Leu

CAG Gln

GTC Val

ATT  Ile#

GAG Glu

GTC Val

316 106

ACA Thr

GCC Ala

TAC Tyr

AAT Asn

CGG  Arg#

GAC Asp

AGC Ser

TTT Phe

GAT Asp

ACC Thr

ACT Thr

CGG Arg

CAG Gln

AGG Arg

CTG Leu

361 121

GTG Val

CTG Leu

GAG Glu

ATT  Ile#

GGG Gly

GAC Asp

CCA Pro

GAA Glu

GGC Gly

CCC Pro

CTG Leu

CTG Leu

CCA Pro

TAC Tyr

CAA Gln

406 136

GCC Ala

GAG  Glu#

TTC Phe

CTG  Leu#

GTG Val

CGC  Arg#

AGC Ser

CAC His

GAT Asp

GCG Ala

GAG Glu

GAG Glu

GTG Val

CTG Leu

CCC Pro

451 151

TCA Ser

ACA Thr

CCT Pro

GCC Ala

AGC Ser

CGC Arg

TTC Phe

CTC  Leu#

TCA Ser

GCC Ala

TTG Leu

GGG Gly

GGA Gly

CTC Leu

TGG Trp

496 166

GAG Glu

CCC Pro

GGA Gly

GAG Glu

CTT Leu

CAG Gln

CTG Leu

CTC  Leu#

AAC Asn

GTC  Val#

ACC Thr

TCT Ser

GCC Ala

TTG Leu

GAC Asp

541 181

CGT  Arg#

GGG Gly

GGC Gly

CGT Arg

GTC Val

CCC Pro

CTT Leu

CCC Pro

ATT Ile

GAG Glu

GGC Gly

CGA Arg

AAA Lys

GAA Glu

GGG  Gly#

586 196

GTA  Val#

TAC Tyr

ATT Ile

AAG Lys

GTG Val

GGT Gly

TCT Ser

GCC Ala

TCA Ser

CCT  Pro#

TTT Phe

TCT Ser

ACT  Thr#

TGC Cys

CTG Leu

631 211

AAG Lys

ATG Met

GTG Val

GCA Ala

TCC  Ser#

CCC Pro

GAT Asp

AGC Ser

CAC His

GCC Ala

CGC  Arg#

TGT Cys

GCC Ala

CAG  Gln#

GGC Gly

676 226

CAG Gln

CCT  Pro#

CCA  Pro#

CTT Leu

CTG Leu

TCT Ser

TGC  Cys#

TAC Tyr

GAC Asp

ACC Thr

TTG Leu

GCA Ala

CCC  Pro#

CAC His

TTC Phe

721 241

CGC Arg

GTT  Val#

GAC Asp

TGG Trp

TGC Cys

AAT Asn

GTG  Val#

ACC Thr

CTG Leu

GTG Val

GAT Asp

AAG Lys

TCA Ser

GTG Val

CCG Pro

766 256

GAG Glu

CCT Pro

GCA Ala

GAT Asp

GAG Glu

GTG Val

CCC  Pro#

ACC Thr

CCA Pro

GGT Gly

GAT Asp

GGG Gly

ATC Ile

CTG Leu

GAG Glu

811 271

CAT His

GAC  Asp#

CCG Pro

TTC Phe

TTC Phe

TGC Cys

CCA Pro

CCC Pro

ACT Thr

GAG Glu

GCC Ala

CCA Pro

GAC Asp

CGT  Arg#

GAC Asp

856 286

TTC Phe

TTG Leu

GTG Val

GAT Asp

GCT Ala

CTG Leu

GTC Val

ACC Thr

CTC Leu

CTG Leu

GTG Val

CCC Pro

CTG Leu

CTG Leu

GTG Val

901 301

GCC Ala

CTG Leu

CTT Leu

CTC Leu

ACC Thr

TTG Leu

CTG Leu

CTG Leu

GCC Ala

TAT Tyr

GTC Val

ATG  Met#

TGC Cys

TGC Cys

CGG Arg

946 316

CGG Arg

GAG Glu

GGA Gly

AGG Arg

CTG Leu

AAG Lys

AGA Arg

GAC Asp

CTG Leu

GCT Ala

ACC Thr

TCC Ser

GAC Asp

ATC Ile

CAG Gln

991 331

ATG Met

GTC Val

CAC His

CAC His

TGC Cys

ACC Thr

ATC Ile

CAC His

GGG Gly

AAC Asn

ACA Thr

GAG Glu

GAG Glu

CTG Leu

CGG Arg

1036 346

CAG Gln

ATG Met

GCG Ala

GCC Ala

AGC Ser

CGC Arg

GAG Glu

GTG Val

CCC Pro

CGG Arg

CCA Pro

CTC Leu

TCC Ser

ACC Thr

CTG Leu

1081 361

CCC Pro

ATG Met

TTC Phe

AAT Asn

GTG Val

CAC His

ACA Thr

GGT Gly

GAG Glu

CGG Arg

CTG Leu

CCT Pro

CCC Pro

CGC Arg

GTG Val

1126 376

GAC Asp

AGC Ser

GCC Ala

CAG Gln

GTG Val

CCC Pro

CTC Leu

ATT Ile

CTG Leu

GAC Asp

CAG Gln

CAC His

TGA Ter

*Note the previously determined missense mutations marked with #. The present deletion was marked with ## and bold letters.

3. Results

The muscle biopsy showed dystrophic changes like contraction, regeneration (Figure 1), degeneration, necrosis, nuclear internalization, and fibrosis. In addition, many pathological immature myofibers were visualized using the neonatal myosin staining (Figure 2). Based on immunostaining, dystrophin and spectrin expressions were normal. Except for isolated deficient fibers, beta (β) sarcoglycan and delta (δ) sarcoglycan were present at normal levels, whereas α-SGC and γ-SGC were diffusely absent (Figure 3).

Figure 1.

Differences in the size and shape of myofibers as well as regeneration (HEx 200).

Figure 2.

Immature pathological fibers visualized with anti-neonatal myosin antibody staining (DABx 100).

Figure 3.

Diffuse absence of sarcolemmal α-SGC (a) and γ-SGC (d) expression and normal β-SGC (b) and δ-SGC (c) expression (DABx 200).

Based on analysis of the proband, we have identified a previously undetermined homozygous 7 bp deletion in exon 3 (Figure 4). A similar heterozygous deletion was found in both parents (Figures 5 and 6). Location of this deletion was also indicated in Table 1. In addition, there were no abnormalities in the dystrophin gene and the other sarcoglycan genes (SGCB, SGCD, and SGCG) in the patient and his parents.

Figure 4.

Proband exon 3 homozygous del TACACCC site.

Figure 5.

Maternal heterozygous del TACACCC site.

Figure 6.

Paternal heterozygous del TACACCC site.

4. Discussion

Human SGCA cDNA from a human skeletal muscle library was isolated and sequenced in 1993. This gene consisted of 10 exons. The protein product of SGCA gene consisted of 387 amino acids with an extracellular N-terminus, a transmembrane domain, and an intracellular C-terminus. Northern blot analysis showed that human adhalin mRNA was expressed at the highest levels in skeletal muscle. It was also expressed in cardiac muscle and in the lung, but at much lower levels. On the contrary, adhalin mRNA was not detected in the brain. It was also reported that the adhalin mRNA from cardiac muscle was shorter relative to skeletal muscle and that the base sequence encoding the transmembrane domain was absent. It is known that LGMD-2D primarily affects skeletal muscles while brain and peripheral nerve functions are largely preserved. Briefly, the less severe cardiac dysfunction and lack of mental retardation in patients with LGMD-2D may be explained by the lower expression of α-SGC in cardiac muscle and the absence of adhalin expression in the brain [1, 3, 10]. In the patient described in this report, we did not find clinical evidence of cardiac involvement, decreased intellectual capacity, or denervation (as demonstrated by electromyography). The course of the disease in this case suggests that this novel deletion may cause a milder phenotype of LGMD-2D despite the diffuse absence of α-SGC and γ-SGC.

Immunohistochemical analysis of sarcolemmal proteins in muscle biopsies like dystrophin, SGCs, merosin, and dysferlin is an important part of the diagnostic evaluation of patients with muscular dystrophy. Reduced or absent sarcolemmal expression of one of the 4 SGCs can be found in patients with any type of LGMDs and also in patients with dystrophinopathies. It has previously been suggested that different patterns of SGC expression could predict the primary genetic defect and that genetic analysis could be directed by these patterns [5–8]. However, Klinge et al. [9] reported that residual SGC expression could be highly variable and an accurate prediction of the genotype could not be achieved. Babameto-Laku et al. [4] also determined that the concomitant absence of α-SGC and γ-SGC expression was caused by defects in the SGCA gene. Therefore, they recommended using antibodies against all four SGCs for immunoanalysis of skeletal muscle sections. Similarly, a concomitant reduction in dystrophin and any of the SGCs may illustrate the importance of considering coexisting dystrophinopathies in patients with sarcoglycan-deficient LGMD [9–13]. 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. However, in the patient described in this report, dystrophinopathies, such as DMD and BMD, were ruled out because the expression of sarcolemmal dystrophin was diffusely present and molecular tests for dystrophin gene were normal.

At present, more than 70 mutations have been reported in the SGCA gene that cause changes in the α-SGC glycoprotein. Approximately a two-thirds of mutations are missense mutations that generate a complete protein with a single residue substitution, whereas other mutations like nucleotide replacements, duplications, deletions, or insertions produce truncated, incomplete, or anomalous proteins. Almost all missense mutations map to the extracellular domain which is a critical region for the organization of SGCs and their association with dystroglycan. Only a single missense mutation maps to the intracellular domain and causes LGMD-2D in homozygous cases. Similarly, two mutations caused by deletions generate a normal extracellular portion of α-SGC and truncated intracellular tails. At present, there is no data about the intracellular tail of the α-SGC protein and its function [1, 10–14]. In the family described in this report, we discovered a novel deletion in the TACACCC site of exon 3 that would cause a frame-shift mutation. The past literature highlights that the prediction of pathological consequences associated with different mutations of SGCA gene is very complex. It is not clear whether this novel deletion generates a severe disease phenotype or whether it also has additional, undetermined consequences.

Patients with any of the LGMDs may be clinically indistinguishable from those with the primary dystrophinopathies. It is likely that the prevalence of LGMD is underestimated and a number of male patients are incorrectly diagnosed with DMD or BMD [13]. A definitive diagnosis rests on performing the appropriate immunohistochemical examination as well as doing a molecular analysis. A normal dystrophin staining pattern should be seen as well as an autosomal recessive mode of inheritance. In contrast, the patients with dystrophinopathies may show variable findings from a normal to a regional absence or a mosaic pattern of sarcolemmal staining with anti-SGCs antibodies which correspond to an abnormal organization of the cell-membrane-associated dystrophin glycoprotein complex. Therefore, it is necessary to perform a careful examination of the immunohistochemical staining as well as a genetic study in order to make the correct diagnosis.

In summary, this report describes a novel deletion that adds to the growing list of defects associated with LGMD-2D and further emphasizes the importance of systematic analysis of all related genes, instead of limiting the analysis to the one SGC gene that is hypothesized to be the cause of the abnormalities. In this study, we also highlight the complexity of staining patterns associated with sarcolemmal proteins and the importance of careful analysis of this staining pattern in order to narrow the differential diagnosis of muscular dystrophies.

Conflict of Interests

The authors declare that there is no conflict of interests.