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

Authors: Gulden Diniz; Hulya Tosun Yildirim; Sarenur Gokben; Gul Serdaroglu; Filiz Hazan; Kanay Yararbas; Ajlan Tukun
Journal: Case Reports in Genetics
 (2014)

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## 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.


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## Body

## 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 Table1.Table 1
Nucleotide and amino acid sequences ofα-SGC gene.
11
ATGMet
GCTAla
GAGGlu
ACAThr
CTCLeu
TTCPhe
TGGTrp
ACTThr
CCTPro
CTCLeu
CTCLeu
GTGVal
GTTVal
CTCLeu
CTGLeu
4616
GCAAla
GGGGly
CTGLeu
GGGGly
GACAsp
ACCThr
GAGGlu
GCCAla
CAGGln
CAGGln
ACCThr
ACGThr
CTALeu
CACHis
CCAPro#
9131
CTTLeu#
GTGVal
GGCGly
CGTArg#
GTCVal
TTTPhe
GTGVal
CACHis
ACCThr
TTGLeu
GACAsp
CATHis
GAGGlu
ACGThr
TTTPhe
13646
CTGLeu
AGCSer
CTTLeu
CCTPro
GAGGlu
CATHis
GTCVal
GCTAla#
GTCVal
CCAPro
CCCPro
GCTAla
GTCVal
CACHis
ATCIle
18161
ACCThr
TACTyr#
CACHis
GCCAla
CACHis
CTCLeu
CAGGln
GGAGly#
CACHis
CCAPro
GACAsp
CTGLeu
CCCPro
CGGArg#
TGGTrp
22676
CTCLeu
CGCArg#
TAC Tyr##
ACC Thr##
CAG Gln##
CGCArg#
AGCSer
CCCPro
CACHis
CACHis
CCTPro
GGCGly
TTCPhe
CTCLeu#
TACTyr#
27191
GGCGly#
TCTSer
GCCAla#
ACCThr
CCAPro
GAAGlu
GATAsp#
CGTArg#
GGGGly
CTCLeu
CAGGln
GTCVal
ATTIle#
GAGGlu
GTCVal
316106
ACAThr
GCCAla
TACTyr
AATAsn
CGGArg#
GACAsp
AGCSer
TTTPhe
GATAsp
ACCThr
ACTThr
CGGArg
CAGGln
AGGArg
CTGLeu
361121
GTGVal
CTGLeu
GAGGlu
ATTIle#
GGGGly
GACAsp
CCAPro
GAAGlu
GGCGly
CCCPro
CTGLeu
CTGLeu
CCAPro
TACTyr
CAAGln
406136
GCCAla
GAGGlu#
TTCPhe
CTGLeu#
GTGVal
CGCArg#
AGCSer
CACHis
GATAsp
GCGAla
GAGGlu
GAGGlu
GTGVal
CTGLeu
CCCPro
451151
TCASer
ACAThr
CCTPro
GCCAla
AGCSer
CGCArg
TTCPhe
CTCLeu#
TCASer
GCCAla
TTGLeu
GGGGly
GGAGly
CTCLeu
TGGTrp
496166
GAGGlu
CCCPro
GGAGly
GAGGlu
CTTLeu
CAGGln
CTGLeu
CTCLeu#
AACAsn
GTCVal#
ACCThr
TCTSer
GCCAla
TTGLeu
GACAsp
541181
CGTArg#
GGGGly
GGCGly
CGTArg
GTCVal
CCCPro
CTTLeu
CCCPro
ATTIle
GAGGlu
GGCGly
CGAArg
AAALys
GAAGlu
GGGGly#
586196
GTAVal#
TACTyr
ATTIle
AAGLys
GTGVal
GGTGly
TCTSer
GCCAla
TCASer
CCTPro#
TTTPhe
TCTSer
ACTThr#
TGCCys
CTGLeu
631211
AAGLys
ATGMet
GTGVal
GCAAla
TCCSer#
CCCPro
GATAsp
AGCSer
CACHis
GCCAla
CGCArg#
TGTCys
GCCAla
CAGGln#
GGCGly
676226
CAGGln
CCTPro#
CCAPro#
CTTLeu
CTGLeu
TCTSer
TGCCys#
TACTyr
GACAsp
ACCThr
TTGLeu
GCAAla
CCCPro#
CACHis
TTCPhe
721241
CGCArg
GTTVal#
GACAsp
TGGTrp
TGCCys
AATAsn
GTGVal#
ACCThr
CTGLeu
GTGVal
GATAsp
AAGLys
TCASer
GTGVal
CCGPro
766256
GAGGlu
CCTPro
GCAAla
GATAsp
GAGGlu
GTGVal
CCCPro#
ACCThr
CCAPro
GGTGly
GATAsp
GGGGly
ATCIle
CTGLeu
GAGGlu
811271
CATHis
GACAsp#
CCGPro
TTCPhe
TTCPhe
TGCCys
CCAPro
CCCPro
ACTThr
GAGGlu
GCCAla
CCAPro
GACAsp
CGTArg#
GACAsp
856286
TTCPhe
TTGLeu
GTGVal
GATAsp
GCTAla
CTGLeu
GTCVal
ACCThr
CTCLeu
CTGLeu
GTGVal
CCCPro
CTGLeu
CTGLeu
GTGVal
901301
GCCAla
CTGLeu
CTTLeu
CTCLeu
ACCThr
TTGLeu
CTGLeu
CTGLeu
GCCAla
TATTyr
GTCVal
ATGMet#
TGCCys
TGCCys
CGGArg
946316
CGGArg
GAGGlu
GGAGly
AGGArg
CTGLeu
AAGLys
AGAArg
GACAsp
CTGLeu
GCTAla
ACCThr
TCCSer
GACAsp
ATCIle
CAGGln
991331
ATGMet
GTCVal
CACHis
CACHis
TGCCys
ACCThr
ATCIle
CACHis
GGGGly
AACAsn
ACAThr
GAGGlu
GAGGlu
CTGLeu
CGGArg
1036346
CAGGln
ATGMet
GCGAla
GCCAla
AGCSer
CGCArg
GAGGlu
GTGVal
CCCPro
CGGArg
CCAPro
CTCLeu
TCCSer
ACCThr
CTGLeu
1081361
CCCPro
ATGMet
TTCPhe
AATAsn
GTGVal
CACHis
ACAThr
GGTGly
GAGGlu
CGGArg
CTGLeu
CCTPro
CCCPro
CGCArg
GTGVal
1126376
GACAsp
AGCSer
GCCAla
CAGGln
GTGVal
CCCPro
CTCLeu
ATTIle
CTGLeu
GACAsp
CAGGln
CACHis
TGATer
*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 (Figure1), 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).Diffuse absence of sarcolemmalα-SGC (a) and γ-SGC (d) expression and normal β-SGC (b) and δ-SGC (c) expression (DABx 200).
(a)
(b)
(c)
(d)Based on analysis of the proband, we have identified a previously undetermined homozygous 7 bp deletion in exon 3 (Figure4). 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 ofα-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 theα-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.



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*Source: 248561-2014-06-22.xml*