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Sarcoglycanopathies: Can muscle immunoanalysis predict the genotype?
Neuromuscular Disorders 18 (2008) 934–941
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Sarcoglycanopathies: Can muscle immunoanalysis predict the genotype? Lars Klinge a, Gabriele Dekomien b, Ahmed Aboumousa a, Richard Charlton a, Jörg T. Epplen b, Rita Barresi a, Kate Bushby a, Volker Straub a,* a b
Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, NE1 3BZ Newcastle-upon-Tyne, UK Ruhr-University Bochum, Human Genetics, Universitätsstraße 150, 44801 Bochum, Germany
a r t i c l e
i n f o
Article history: Received 9 June 2008 Received in revised form 24 July 2008 Accepted 16 August 2008
Keywords: Muscular dystrophy Sarcoglycanopathy Sarcoglycans Dystroglycan Immunoanalysis
a b s t r a c t Muscle immunoanalysis of the sarcoglycan complex is an important part of the diagnostic evaluation of muscle biopsies in patients with autosomal recessive limb-girdle muscular dystrophy. Reduced or absent sarcolemmal expression of one or all of the four sarcoglycans (a-, b-, c-, d-sarcoglycan) can be found in patients with limb-girdle muscular dystrophy 2C-F (LGMD2C-F) and also in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). It has previously been suggested that different patterns of sarcoglycan expression could predict the primary genetic defect, and that genetic analysis could be directed by these patterns. In this first UK study we studied 24 genetically characterized patients with sarcoglycan deficient LGMD, in 22 of whom muscle immunoanalysis data were available. Thirteen patients showed asarcoglycan deficient LGMD2D, 7 patients b-sarcoglycan deficient LGMD2E, 3 patients c-sarcoglycan deficient LGDM2C, and one patient d-sarcoglycan deficient LGMD2F. Muscle biopsies were analysed in one centre without knowledge of the established genetic diagnosis. Our results demonstrated that residual sarcoglycan expression is highly variable and does not enable an accurate prediction of the genotype. Considering previous reports of sarcoglycanopathy patients with an isolated loss of one sarcoglycan we recommend to use antibodies against all four sarcoglycans for immunoanalysis of skeletal muscle sections. A concomitant reduction of dystrophin and b-dystroglycan was observed more frequently than previously reported and illustrates the important differential diagnosis of DMD and BMD for sarcoglycan deficient LGMD. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction The autosomal recessive limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of disorders leading to progressive muscle wasting and weakness, with evidence for at least 14 distinct loci at present (http://www.musclegenetable.org). Four of these (LGMD2C-F) are caused by mutations in the genes coding for the sarcoglycan proteins a-, b-, c-, and d-sarcoglycan (collectively also referred to as sarcoglycanopathies) [1–5]. Two further sarcoglycans have also been identified (e-, and f-sarcoglycan [6– 8]). c-, d-, and f-sarcoglycan share significant sequence similarity while a-sarcoglycan is highly related to e-sarcoglycan. Mutations in the e-sarcoglycan gene are associated with myoclonus dystonia syndrome in humans [9], while no disease has so far been associated with mutations in the f-sarcoglycan gene. In skeletal and cardiac muscle a-, b-, c-, and d-sarcoglycan assemble into a tetrameric complex forming a subcomplex within the dystrophin glycoprotein complex (DGC). The sarcoglycans are glycosylated proteins with single transmembrane domains [10], and correct assembly of the * Corresponding author. Tel.: +44 1912418655; fax: +44 1912418770. E-mail address: [email protected] (V. Straub). 0960-8966/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.08.003
sarcoglycan complex is required for maintenance of the sarcolemma [11–13]. Their function is not completely understood but they appear to have both mechanical and non-mechanical roles that mediate interactions between the extracellular matrix, the sarcolemma and the cytoskeleton [14,15]. b-, d-, e-, and f-sarcoglycan have also been found to be important for myelin stability in Schwann cells [16]. Mutations in the gene for a-sarcoglycan are generally the most common sarcoglycan mutations, whereas mutations in d-sarcoglycan are the rarest. Due to a founder mutation in the c-sarcoglycan gene LGMD2C is the most common sarcoglycanopathy in the North African population [17]. In order to reach a diagnosis and provide accurate counselling for patients affected by sarcoglycanopathy, it is necessary to identify which sarcoglycan protein is primarily affected. So far, this can only be achieved through mutation analysis. Muscle immunoanalysis with antibodies against a-, b-, c-, and d-sarcoglycan is part of the diagnostic work-up of patients with autosomal recessive LGMD. However, a deficiency of any single component of the sarcoglycan protein complex may lead to concomitant reduction or loss of the other sarcoglycans [10,11,18–20]. Therefore, it would be of great advantage to identify expression patterns that could help to develop a diagnostic algorithm for the consecutive molecular
L. Klinge et al. / Neuromuscular Disorders 18 (2008) 934–941
analysis of the four sarcoglycan genes. Some studies have been published showing distinct patterns of sarcoglycan expression/ labelling [10,11,18–20], but so far data are not entirely consistent necessitating further investigation [19]. In order to further explore the feasibility of a diagnostic algorithm to direct sarcoglycan gene mutation analysis, we analysed clinical, biochemical, and molecular findings in a cohort of 24 patients with sarcoglycanopathy from the UK. The main aim of this study was to investigate if muscle immunoanalysis could correctly predict which of the four sarcoglycan genes was primarily affected in these patients.
2. Materials and methods We present data of 24 patients with sarcoglycanopathy studied at the Newcastle muscle centre, UK. These patients originated from 20 different families (13 patients with a-sarcoglycanopathy from 10 families, 7 patients with b-sarcoglycanopathy from 6 families, 3 patients with c-sarcoglycanopathy from 3 families and one patient with d-sarcoglycanopathy). From this cohort muscle biopsies were available for 22 patients. The clinical history of the patients was reviewed, and muscle sections and Western blots were analysed and rated by three independent investigators, all experienced in the interpretation of muscle immunoanalysis. Biopsy material was not suitable/not interpretable for immunohistochemistry in 3 patients, and only Western blots were analysed. No Western blot was available in one patient.
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1:100 in 0.1 M lysine in 40% Foetal Calf Serum. Sections were washed, visualised with diaminobenzidine tetrahydrochloride (DAB) and counterstained with Carazzi’s haematoxylin prior to dehydration and mounting. Control sections were labelled without primary antibodies, and all sections were compared with samples with non-LGMD neuromuscular disorders, and with normal muscle. 2.3. Multiplex immunoanalysis on Western blots Multiplex immunoanalysis on Western blots from biphasic polyacrylamide gels (4–12% gradient) was performed using antibodies to dysferlin (NCL-hamlet exon 53), dystrophin (Dy8/6C5 C-terminus and Dy4/6D3 rod domain), a-sarcoglycan, b-dystroglycan, c-sarcoglycan, calpain 3 (NCL-CALP-2C4 and NCL-CALP-12A2), and caveolin 3 (BD610420). Myosin heavy chain staining on the post-blotted gel was used as loading control. 2.3.1. Evaluation of biopsies Three independent evaluators, all experienced in reading of muscle biopsies and Western blots, evaluated the biopsies and were blinded as to the underlying genotype of the patients. Protein expression on sections and blots was scored into normal +++, slight reduction ++, reduction +, severe reduction +/ , and absence . b-Spectrin was used as a marker of membrane integrity on immunohistochemistry. Based on the expression pattern of the sarcoglycans and other proteins of the DGC, the evaluators made a prediction of the underlying genotype of the patients.
2.1. Mutation analysis Screening for mutations was performed by polymerase chain reaction (PCR) for all exons of the four sarcoglycan (SGC) genes (SGCA, 9 exons; SGCB, 6 exons; SGCG, 8 exons; SGCD, 8 exons) by denaturating high performance liquid chromatography (DHPLC; transgenomic Cheshire, UK; analysis software Wavemaker 4.1). DNA samples with deviating melting curves were directly sequenced (MegaBace1000 Amersham). In order to exclude intragenic deletions multiplex ligation-dependent probe amplification (MLPA) was applied in all patients with homozygous mutations and in patients with only a single mutation detected by DHPLC analysis.
3. Results 3.1. Mutation analysis Mutation analysis showed the following distribution of sarcoglycan mutations either in homozygous or compound heterozygous status in our cohort: a-sarcoglycan 54%, b-sarcoglycan 29%, c-sarcoglycan 13%, and d-sarcoglycan 4%. Of these mutations 11 were novel. The mutations and the predicted molecular consequences are listed in Table 1. The 3 LGMD2C patients in our cohort all had homozygous missense mutations.
2.2. Muscle biopsy Immunoanalysis 3.2. Clinical information Muscle biopsies were analysed as part of the National Commissioning Group (NCG) designated specialised diagnostic service for Limb-Girdle Muscular Dystrophies (LGMD). Optimised immunohistochemical and multiplex Western blot protocols were used for the demonstration and resolution of muscular dystrophy associated proteins as previously described [21]. Open or needle biopsies of muscle were snap frozen in isopentane cooled in liquid nitrogen, mounted in OCT and 6lm sections were cut and mounted onto SuperFrostÒ Plus slides. Immunolabelling was carried out using a standard protocol. Briefly, sections were equilibrated to room temperature and washed for 15 min in PBS pH 7.3 containing 0.1% Triton X to permeabilise the membranes. Excess buffer was removed and sections incubated in optimally diluted primary antibody to b-spectrin (RBC2/3D5), b-dystroglycan (43DAG/8D5), C-terminus dystrophin (Dy8/6C5) and N-terminus dystrophin (Dy10/12B2), a-sarcoglycan (Ad1/20A6), b-sarcoglycan (1/5B1), c-sarcoglycan (35DAG/ 21B5), d-sarcoglycan (3/12C1) (all from Novocastra). All antibodies were diluted in 40% Foetal Calf Serum containing 0.1 M lysine and incubated overnight at 4 °C. Following 2 10 min washes in PBS/Triton sections were incubated for 90 min at room temperature in HRP conjugated rabbit anti mouse Ig (Dako P260) diluted
The clinical details of the patients are listed in Table 2. All patients showed limb-girdle weakness, calf hypertrophy and elevated serum creatine kinase levels (4–100 times the normal value). Age of onset varied from 1.5 up to 30 years. The phenotype was graded into mild, intermediate and severe depending on the age of onset and progression of the disease. Mean age of onset in a-sarcoglycanopathy was 13.2 ± 2.3 years, 6.5 ± 1.1 years in b-sarcoglycanopathy, and 6.7 ± 1.4 years in c-sarcoglycanopathy. Overall, the progression of symptoms in patients with a-sarcoglycanopathy was diverse: 4 patients lost ambulation before the age of 16 years while nine patients are still ambulant, 3 of them aged 50 years and above. Patients with b-sarcoglycanopathy appeared to display a slightly more consistent and severe phenotype with 4/7 patients having lost ambulation before the age of 25 years. Echocardiography data were available in 21 patients. The mean age at which echocardiography was performed was 22.3 years. None of the patients showed evidence of cardiomyopathy. Two patients (one affected by a-, and one by b-sarcoglycanopathy) needed nocturnal ventilator support from the age of 26 and 37 years on.
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Table 1 Molecular data of the patients No.
Allele 1
Allele 2
Type
Mutation
Location of mutation allele 1
Location of mutation allele 2
1 2a
c.695G>C(p.Cys232Ser) c.347_351dupCTCGG (Gln118LeufsX94)
c.695TG>C(p.Cys232Ser) c.371C>T(p.Ile124Thr)
New
Extracellular Extracellular
Extracellular Extracellular
2b
c.347_351dupCTCGG (Gln118LeufsX94)
c.371C>T(p.Ile124Thr)
Extracellular
Extracellular
3
c.347_351dupCTCGG (Gln118LeufsX94)
c.229C>T(p.Arg77Cys)
Extracellular
Extracellular
4 5
c.292C>A (p.Arg98Ser) c.229C>T(p.Arg77Cys)
c.292C>A(p.Arg98Ser) c.739G>A(p.Val247Met)
Extracellular Extracellular
Extracellular Extracellular
6a
c.229C>T(p.Arg77Cys)
c.739G>A(p.Val247Met)
Extracellular
Extracellular
6b
c.229C>T(p.Arg77Cys)
c.739G>A(p.Val247Met)
Extracellular
Extracellular
7
c.371C>T (p.Ile124Thr)
c.739G>A(p.Val247Met)
Extracellular
Extracellular
8
c.613C>T(p.Pro205Ser)
c.265C>T(p.Leu89Phe)
Missense Frameshift/ missense Frameshift/ missense Frameshift/ missense Missense Missense/ missense Missense/ missense Missense/ missense Missense/ missense Missense
Extracellular
Extracellular
9a
c.371C>T (p.Ile124Thr)
c.850C>T (p.Arg284Cys)
Extracellular
Extracellular
9b
c.371C>T (p.Ile124Thr)
c.850C>T (p.Arg284Cys)
Extracellular
Extracellular
10
c.293C>A (p.Arg98His) and in c: SGC exon 8 c.778_779del AGSer360ProfsX57)
488insGLeu164ThrfsX27
New, allele 2
Extracellular
Extracellular
11
IVS2+5G>A
IVS2+5G>A
New
Intracellular
Intracellular
12a 12b 13
c.23_54 ins c.23_54 ins, exon 1 c.1_2delAT
c.23_54 ins c.23_54 ins c.341C>T(p.Ser114Phe)
New New New
Intracellular Intracellular Intracellular
Intracellular Intracellular Extracellular
14
exon 1 c.1_2delAT
c.341C>T(p.Ser114Phe)
New
Intracellular
Extracellular
15 16
c.341C>T(p.Ser114Phe) c.341C>T(Ser114Phe)
c.341C>T(p.Ser114Phe)
Extracellular Extracellular
Extracellular
LGMD2C (csarcoglycan)
17 18 19
c.244G>A (p.Gly82Arg) c.158T>C (p.Leu53Pro) c.158T>C (p.Leu53Pro)
c.244G>A (p.Gly82Arg) c.158T>C (p.Leu53Pro) c.158T>C (p.Leu53Pro)
Missense Missense Missense
New New New
Extracellular TM TM
Extracellular TM TM
LGMD2F (dsarcoglycan)
20
c.226G>T (p.Gly76Cys)
c.226G>T (p.Gly76Cys)
Missense
New
Extracellular
Extracellular
LGMD2D (asarcoglycan)
LGMD2E (bsarcoglycan)
Missense/ missense Missense/ missense Missense/ frameshift Splice mutation Duplication Duplication Frameshift/ missense Frameshift/ missense Missense Missense
New
Both new
No., patient number; TM, transmembrane.
Table 2 Clinical data of the patients No.
Age at onset (years)
Functional state
1 2a 2b 3 4 5 6a 6b 7 8 9a 9b 10
6 8 7 4 5 30 18 15 15 28 14 14 7
Lost ambulation Lost ambulation Lost ambulation Lost ambulation Climb stairs 6 Ambulant at 50 Ambulant at 56 Ambulant at 50 Ambulant at 26 Ambulant at 34 Ambulant at 40 Ambulant at 38 Climb stairs 10
11 12a 12b 13 14 15 16
6 7 10 8 1.5 9 4
LGMD2C
17 18 19
LGMD2F
20
LGMD2D
LGMD2E
Cardiac.
Resp.
Skel. deform.
CK
Age (years) at CK
n.a. No No No No No n.a. n.a. No No No No No
n.a. No No Yes No No n.a. n.a. No No No No No
n.a. No No Scoliosis No No n.a. n.a. No No No No No
15.77 5000 2500 High 2736 2000 1160 n.a. 5296 1900 6000 1700 12.297
7 14 16 n.a. 5 50 56
Lost ambulation at 11 Ambulant at 28 Ambulant at 25 Lost ambulation at 25 Lost ambulation at 15 Lost ambulation at 14 Occasional wheelchair at 11
No No No No No No No
No No No Yes No No No
No No No Scoliosis No Scoliosis No
800 2000 3358 8812 4640 7000 2300
3 8 9
With aid at 8y With aid at 30 Ambulant at 11
No No n.a.
No No n.a.
No Scoliosis n.a.
19.244 950 n.a.
7 21
5
Lost ambulation at 12
n.a.
n.a.
n.a.
19,000
12
at at at at
7 10 10 15
21 32 31 26 10 26 23 12 9 7
No., patient number, n.a., not available, yrs., years; Cardiac, cardiac involvement; resp., respiratory involvement, skel. deform., skeletal deformities; CK, serum Creatin Kinase.
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L. Klinge et al. / Neuromuscular Disorders 18 (2008) 934–941 Table 3 Muscle immunoanalysis data Immunohistochemistry
LGMD2D
LGMD2E
LGMD2C
LGMD2F
No.
Phenotype
a-SG
1 2a 3 4 5 6a 6b 7 8 9a 10
Severe Severe Severe Severe Mild Mild Mild Intermediate Mild Mild Severe
+
11 12a 12b 13 14 15 16
Severe Intermediate Intermediate Severe Severe Severe Severe
17 18 19
Severe Intermediate Too young to judge
20
Severe
c-SG
b-SG
d-SG
b-Spectrin
b-DG
Dys-N
Dys-C
a-SG
++ + + +/
++ + + +/
+ +++ +++ +++
+ ++ +++ +
++ +++ +++ ++
++ +++ +++ ++
+/
+
++
+
+++
++
++
+++
+ +/ +
++ + + +/
++ + + +/
+++ +++ +++ +++
+++ ++ +++ +
+++ ++ +++ ++
+++ +++ +++ ++
+/ +/ + ++ + + + +/
+/ +/ + +/-
+ +
+/ + + + + n.a. +/
++ +++ +++ ++ +++ ++ ++
+ + + +++ + + +/
+++ +++ ++
+++ +++ +++
+ +/
++ ++ +++
+++ +++ +++
+/ + ++
++ ++ +++
+/ ++ ++
+ + ++
++ +++ +++
++
+/
++
++
+/
Not suitable + Not suitable + + +
+ + Not suitable + +
+ +
Western Blot
+ n.a.
+ +/
+++, normal; ++, slightly reduced; +, reduced; +/ , severely reduced;
+/n.a.
c-SG
+/
+ +
-
+ + +/
b-DG
Dys-N
Dys-C
Predicted diagnosis
++ ++ ++ ++ + ++ ++
+++ +++ +++ +++ ++ +++ ++ +++ +++ +++ +++
+++ +++ +++ +++ ++ +++ ++ +++ +++ +++ +++
LGMD2C LGMD2D Impossible Impossible Impossible LGMD2C Impossible LGMD2C LGMD2C Impossible Impossible
+ +/
++ +++ ++
++ +++ +++
+
++
++
Impossible Impossible LGMD2C LGMD2C LGMD2C
++ +++ ++
+/
+
+
LGMD2D
++ ++ ++
+ ++ +++
++ +++ +++
LGMD2C LGMD2C LGMD2C
+
++
++
LGMD2D
, absent; No., patient number; SG, sarcoglycan; b-DG, b-dystroglycan; Dys, dystrophin.
3.3. Correlation of residual sarcoglycan expression, mutation and clinical phenotype Nine patients with a-sarcoglycanopathy showing an intermediate or mild phenotype retained some residual expression of a-sarcoglycan. In those patients displaying a severe phenotype 4 out of 5 showed complete absence of a-sarcoglycan indicating a possible relation of clinical severity and residual expression of a-sarcoglycan in LGMD2D. All patients with residual staining for a-sarcoglycan had missense mutations. This is in accordance with the observation that missense mutations seem to be associated with a milder phenotype while truncating mutations tend to result in a more severe course [22,23]. In b-sarcoglycanopathy 2 patients showed a severe phenotype, 2 of whom had complete loss of b-sarcoglycan staining. One of these patients had a missense and the other one a splice site mutation. The patient with a severe phenotype and residual b-sarcoglycan expression was compound heterozygous with a frameshift mutation on one and a missense mutation on the other allele. In contrast to the patients with LGMD2D all of the LGMD2C patients in our cohort had missense mutations and complete absence of c-sarcoglycan on sections. In the patient with d-sarcoglycanopathy a missense mutation led to a complete absence of the sarcoglycan complex on sections. 3.4. Muscle immunoanalysis (Table 3) The sarcoglycans analysed by Multiplex Western blot were a-sarcoglycan (50 kDa) and c-sarcoglycan (35 kDa). Due to overlap or proximity in molecular size analysis of b-sarcoglycan (43 kDa) and d-sarcoglycan (35 kDa) was not possible within the multiplex method. 3.5. LGMD2D 3.5.1. Immunohistochemistry In LGMD2D patients a-sarcoglycan was absent in 4 out of 9 patients and the most severely reduced protein in one patient. Only
in this 1 patient a correct prediction of the primary defect was made. In 4 patients, c-sarcoglycan was completely absent. In 2 patients, c-sarcoglycan was more severely reduced than a-sarcoglycan. In all LGMD2D patients, it could be noted that b- and dsarcoglycan showed a reduced but preserved expression. b-Dystroglycan was reduced in six patients, while dystrophin was reduced in 5 patients. 3.5.2. Western blot analysis a-Sarcoglycan was absent in 2 out of 11 patients, severely reduced in 4 patients, slightly reduced in one patient. The expression of c-sarcoglycan was affected in all patients: absent in 8, severely reduced in one, and reduced in 2 patients. In 8 patients, the reduction of c-sarcoglycan was more pronounced than the one of the primarily involved a-sarcoglycan. b-Dystroglycan was similarly reduced in all but one case, whereas dystrophin was reduced in 2 patients. 3.6. LGMD2E 3.6.1. Immunohistochemistry In LGMD2E patients b-sarcoglycan was absent in 2 patients. Here a- and c-sarcoglycan were also completely absent and dsarcoglycan was partially preserved. In those 3 patients with low residual expression of b-sarcoglycan c-sarcoglycan was found to be more severely reduced than b-sarcoglycan, and d-sarcoglycan showed a similar expression as b-sarcoglycan. b-Dystroglycan was overall more severely reduced than in a- or c-sarcoglycanopathy, and dystrophin was slightly reduced in three cases. 3.6.2. Western Blot analysis In LGMD2E patients a-sarcoglycan was absent in 2, severely reduced in 2 and reduced in 2 patients. c-Sarcoglycan was absent in all patients. b-Dystroglycan was absent in 1 patient, severely reduced in 2 patients, and reduced in 3 patients. Dystrophin was affected in all but 1 patient.
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3.7. LGMD2C 3.7.1. Immunohistochemistry c-Sarcoglycan was the protein most often and most severely reduced across the whole spectrum of sarcoglycanopathies. In LGMD2C patients, c-sarcoglycan was absent in 3 of 3 patients. In 2 patients, there was partial preservation of the other sarcoglycans. In 1 patient with c-sarcoglycanopathy also a-, and b-sarcoglycan were completely absent with partial preservation of d-sarcoglycan. b-Dystroglycan and dystrophin expression were compromised in all 3 patients. 3.7.2. Western Blot In LGMD2C patients, a-sarcoglycan was reduced in 2 and absent in the third patient, whereas c-sarcoglycan was absent in all patients. b-Dystroglycan was affected in all patients, and dystrophin was affected in 2 patients.
can was also reduced, and reduction of the N-terminal dystrophin domain was more pronounced than that of the C-terminal domain. Overall, Western blot results showed clear reduction of the sarcoglycans and of other DGC components, but the high variability within this pattern did not allow prediction of the primary sarcoglycan gene involved. The sensitivity of the antibody against c-sarcoglycan is slightly lower on Western blots than the antibody against a-sarcoglycan which could contribute to the fact that csarcoglycan is more often and more pronounced reduced on Western blot. This characteristic may be helpful in detecting subtle changes in the expression pattern of c-sarcoglycan. A correct prediction of the mutated gene by the investigators who analysed the biopsy samples was possible in 19%, the prediction was ambiguous in 38%, and wrong in 43% of our samples. Therefore muscle immunoanalysis did not accurately predict the primary defect in the majority of sarcoglycanopathy patients. 4. Discussion
3.8. LGMD2F 3.8.1. Immunohistochemistry Our data on d-sarcoglycanopathy are based on observations in 1 patient and therefore of limited significance. In this patient all sarcoglycans were absent, b-dystroglycan was severely reduced and dystrophin slightly reduced. 3.8.2. Western Blot Absence of a-sarcoglycan, a severe reduction of c-sarcoglycan, a reduction of b-dystroglycan and slight reduction of dystrophin were observed in this patient. 3.9. Prediction of sarcoglycan primarily involved, based on immunohistochemistry and Western blot In 100% of cases, all of the compounds of the sarcoglycan complex were reduced on immunohistochemistry, but our data did not allow the identification of specific expression patterns relating to the primary gene involved, see Table 3. On Western blotting aand c-sarcoglycan were reduced in expression across all four sarcoglycanopathies. The majority of samples showed an additional reduction in the expression of other DGC proteins, especially of b-dystroglycan (16/ 19 cases on immunohistochemistry and 20/21 on Western blot). This reduction was less pronounced in LGMD2D (6/9 cases on immunohistochemistry) and most apparent in LGMD2E patients. In accordance with the biochemical structure of the DGC, dystrophin only showed a reduced expression pattern when b-dystrogly-
We present biochemical data on 22 and clinical and genetic data on 24 patients in the first UK study on genetically proven sarcoglycanopathy. In these patients, we were able to identify 11 novel mutations (7 missense, 2 frame shift, 1 splice site mutation and one duplication, Table 1). Comprehensive muscle immunoanalysis in our cohort of patients unequivocally showed a reduction of all four sarcoglycans and other proteins of the DGC, but no specific expression patterns did emerge which would help to establish an algorithm for mutation analysis (Table 3). Specific patterns of abnormal sarcoglycan expression have on the other hand previously been described in sarcoglycanopathies, especially in LGMD2C. It was suggested that patients with LGMD2C show a significantly reduced or absent staining of c-sarcoglycan with reduced but partly preserved staining of the other sarcoglycan proteins, in particular a- and d-sarcoglycan [12,19,20,22,24– 31]. We found a similar pattern in our patients with c-sarcoglycanopathy, but intriguingly this pattern was not specific for LGMD2C as it was also found in some of our patients with a- and b-sarcoglycanopathy and contributed to the fact that 7 of these patients were misdiagnosed as LGMD2C. Therefore, this staining pattern may be more variable and unspecific across the different forms of sarcoglycanopathy than previously anticipated, especially in those cases with some residual expression of c-sarcoglycan (Table 3). The inconsistent staining pattern in LGMD2C is also illustrated by a patient who showed normal expression of a-, b-, and d-sarcoglycan [32]. Complete maintenance of the other sarcoglycans despite the loss of d-sarcoglycan has also been shown in a patient with LGMD2F [33].
Fig. 1. Different patterns of muscle immunohistochemistry findings in 2 patients affected by a-sarcoglycanopathy (both with missense mutations in the extracellular domain). The insets represent labelling of control muscle. Patient 6a shows a more severe phenotype than patient no. 4. Magnification 20.
L. Klinge et al. / Neuromuscular Disorders 18 (2008) 934–941
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Fig. 2. Muscle immunoanalysis in a patient with Becker muscular dystrophy due to a duplication in the dystrophin gene: immunohistochemistry shows normal dystrophin labelling (antibody directed against the C-terminus) and a reduction of a-sarcoglycan and b-dystroglycan staining. Western blot reveals the increase in molecular weight of dystrophin and, in contrast to immunohistochemistry, demonstrates normal expression of a-sarcoglycan and c-sarcoglycan. Magnification 10.
While some studies have found a-sarcoglycan to be most severely reduced in primary a-sarcoglycanopathy [20,23,24, 30,34,35], other investigations demonstrated a more variable expression pattern from total absence of the entire sarcoglycan complex [22], to some preservation of c-sarcoglycan only [22,36] and to a similar reduction of a- and b-sarcoglycan [36]. In contrast, another study found that severe reduction of a- and b-sarcoglycan did not occur together at all [37]. This inconsistent pattern of sarco-
glycan staining was also evident in our study (Fig. 1). In our cohort of 11 LGMD2D patients, we were able to correctly predict the genotype in only one patient in whom a-sarcoglycan was most reduced on sections and absent on Western blotting. In 6 patients, it was impossible to predict the genotype because c-sarcoglycan was similarly reduced or absent on sections and blots, while b- and d-sarcoglycan were relatively well preserved. In these 6 patients bdystroglycan was best preserved, suggesting and important role
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for b- and d-sarcoglycan to maintain integrity of the DGC. No correlation was found between residual expression of a-sarcoglycan and the site of mutation. Data on the immunoanalysis profile in b-sarcoglycanopathy are also not entirely consistent. Three studies have found the whole sarcoglycan complex to be absent in LGMD2E [22,24,38], one study found near absence or absence of all four sarcoglycans [20], and one study found some residual expression of c-sarcoglycan only [36] (27 patients in total). In contrast, in our group of 6 LGMD2E patients we found residual expression of some of the sarcoglycans in all of them but were unable to predict the genotype correctly in any of them. This was mainly attributed to the fact that a- and c-sarcoglycan were similarly or more reduced than b-sarcoglycan leading to a wrong prediction in four, and making a prediction impossible in two cases. In LGMD2E b-dystroglycan was more markedly reduced than in LGMD2D, again suggesting a closer association between bsarcoglycan and other components of the DGC. Reduction of b-dystroglycan on immunofluorescence has previously been reported in 63% of patients with sarcoglycanopathy [22]. There has also been a report of a reduction of a-dystroglycan in a patient with LGMD2E [39]. We found b-dystroglycan to be reduced in 84% of our patients on immunohistochemistry and in 95% on Western blot. Dystrophinopathy is an important differential diagnosis in patients with sarcoglycanopathy. Immunohistochemical analysis of muscle sections alone can be misleading, as depicted in Fig. 2 in a patient with a duplication in the dystrophin gene. In contrast to immunohistochemistry, which showed normal dystrophin and patchy sarcoglycan and b-dystroglycan staining, Western blot analysis revealed normal expression of a- and c-sarcoglycan and an altered band for dystrophin. In addition, labelling of sections with nNOS and utrophin may provide useful information in some cases as well. Cardiac involvement is a known complication in patients with sarcoglycanopathy. In a study by Politano et al. 1 of 3 LGMD2D patients, 2 of 2 LGMD2E patients, 6 of 9 LGMD2C patients, and 2 of 2 LGMD2F patients had evidence of heart involvement. Seven of these 11 affected patients were presymptomatic [40]. In contrast, cardiac findings in our patients are similar to a recent study on 24 sarcoglycanopathy patients in whom cardiac abnormalities were only found in 1 patient [41]. This suggests that cardiac involvement in sarcoglycanopathy may be less common than previously anticipated, even in LGMD2F. Although predictive patterns of residual sarcoglycan expression may be present in some patients, we conclude that generally sarcoglycan expression may vary significantly in patients with sarcoglycanopathy. Therefore, we recommend labelling of patient biopsies with all four sarcoglycan antibodies in order not to miss a possible isolated loss of any one of the sarcoglycans, as can be the case in some patients [32,33]. As muscle immunoanalysis cannot accurately predict the genotype, genetic analysis should be guided by the known prevalence patterns for LGMD2C, 2D, 2E, and 2F. Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft (KL 1868/1-1 to L.K.), G.D., V.S., H.L., J.T.E., and L.K. are members of the German Muscular Dystrophy Network (MD-NET 01GM0601) funded by the German Ministry of Education and Research (BMBF, Bonn, Germany); http://www.md-net.org. MD-NET is a partner of TREAT-NMD (EC, 6th FP, proposal # 036825; http://www.treat-nmd.eu). We are thankful to Louise Anderson for all her contributions to this study. References [1] Bonnemann CG, Modi R, Noguchi S, et al. Beta-sarcoglycan (A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex. Nat Genet 1995;11:266–73.
[2] Lim LE, Duclos F, Broux O, et al. Beta-sarcoglycan: characterization and role in limb-girdle muscular dystrophy linked to 4q12. Nat Genet 1995;11:257–65. [3] Noguchi S, McNally EM, Ben Othmane K, et al. Mutations in the dystrophinassociated protein gamma-sarcoglycan in chromosome 13 muscular dystrophy. Science 1995;270:819–22. [4] Roberds SL, Leturcq F, Allamand V, et al. Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy. Cell 1994;78:625–33. [5] Nigro V, de Sa Moreira E, Piluso G, et al. Autosomal recessive limb-girdle muscular dystrophy, LGMD2F, is caused by a mutation in the delta-sarcoglycan gene. Nat Genet 1996;14:195–8. [6] Ettinger AJ, Feng G, Sanes JR. epsilon-Sarcoglycan, a broadly expressed homologue of the gene mutated in limb-girdle muscular dystrophy 2D. J Biol Chem 1997;272:32534–8. [7] Kutzick C, Herrmann R, Neumann V, et al. Identification of a novel member of the sarcoglycan complex, zeta-sarcoglycan. Neuromuscul Disord 2001;11:650. [8] Wheeler MT, Zarnegar S, McNally EM. Zeta-sarcoglycan, a novel component of the sarcoglycan complex, is reduced in muscular dystrophy. Hum Mol Genet 2002;11:2147–54. [9] Zimprich A, Grabowski M, Asmus F, et al. Mutations in the gene encoding epsilon-sarcoglycan cause myoclonus-dystonia syndrome. Nat Genet 2001;29:66–9. [10] Ozawa E, Noguchi S, Mizuno Y, et al. From dystrophinopathy to sarcoglycanopathy: evolution of a concept of muscular dystrophy. Muscle Nerve 1998;21:421–38. [11] Holt KH, Campbell KP. Assembly of the sarcoglycan complex Insights for muscular dystrophy. J Biol Chem 1998;273:34667–70. [12] Hack AA, Lam MY, Cordier L, et al. Differential requirement for individual sarcoglycans and dystrophin in the assembly and function of the dystrophin– glycoprotein complex. J Cell Sci 2000;113(Pt 14):2535–44. [13] Straub V, Duclos F, Venzke DP, et al. Molecular pathogenesis of muscle degeneration in the delta-sarcoglycan-deficient hamster. Am J Pathol 1998;153:1623–30. [14] Ozawa E, Mizuno Y, Hagiwara Y, et al. Molecular and cell biology of the sarcoglycan complex. Muscle Nerve 2005;32:563–76. [15] Hack AA, Cordier L, Shoturma DI, et al. Muscle degeneration without mechanical injury in sarcoglycan deficiency. Proc Natl Acad Sci USA 1999;96:10723–8. [16] Cai H, Erdman RA, Zweier L, et al. The sarcoglycan complex in Schwann cells and its role in myelin stability. Exp Neurol 2007;205:257–69. [17] Dubowitz V, Sewry C. Muscle biopsy: a practical approach. Saunders Elsevier; 2007. [18] Bushby KM. The limb-girdle muscular dystrophies-multiple genes, multiple mechanisms. Hum Mol Genet 1999;8:1875–82. [19] Bonnemann CG, Wong J, Jones KJ, et al. Primary gamma-sarcoglycanopathy (LGMD 2C): broadening of the mutational spectrum guided by the immunohistochemical profile. Neuromuscul Disord 2002;12:273–80. [20] Moore SA, Shilling CJ, Westra S, et al. Limb-girdle muscular dystrophy in the United States. J Neuropathol Exp Neurol 2006;65:995–1003. [21] Anderson LV, Davison K. Multiplex Western blotting system for the analysis of muscular dystrophy proteins. Am J Pathol 1999;154:1017–22. [22] Vainzof M, Passos-Bueno MR, Canovas M, et al. The sarcoglycan complex in the six autosomal recessive limb-girdle muscular dystrophies. Hum Mol Genet 1996;5:1963–9. [23] Eymard B, Romero NB, Leturcq F, et al. Primary adhalinopathy (alphasarcoglycanopathy): clinical, pathologic, and genetic correlation in 20 patients with autosomal recessive muscular dystrophy. Neurology 1997;48:1227–34. [24] Draviam R, Billington L, Senchak A, et al. Confocal analysis of the dystrophin protein complex in muscular dystrophy. Muscle Nerve 2001;24:262–72. [25] Hack AA, Ly CT, Jiang F, et al. Gamma-sarcoglycan deficiency leads to muscle membrane defects and apoptosis independent of dystrophin. J Cell Biol 1998;142:1279–87. [26] Takano A, Bonnemann CG, Honda H, et al. Intrafamilial phenotypic variation in limb-girdle muscular dystrophy type 2C with compound heterozygous mutations. Muscle Nerve 2000;23:807–10. [27] Vorgerd M, Gencik M, Mortier J, et al. Isolated loss of gamma-sarcoglycan: diagnostic implications in autosomal recessive limb-girdle muscular dystrophies. Muscle Nerve 2001;24:421–4. [28] McNally EM, Duggan D, Gorospe JR, et al. Mutations that disrupt the carboxylterminus of gamma-sarcoglycan cause muscular dystrophy. Hum Mol Genet 1996;5:1841–7. [29] Sewry CA, Taylor J, Anderson LV, et al. Abnormalities in alpha-, beta- and gamma-sarcoglycan in patients with limb-girdle muscular dystrophy. Neuromuscul Disord 1996;6:467–74. [30] Higuchi I, Kawai H, Umaki Y, et al. Different manners of sarcoglycan expression in genetically proven alpha-sarcoglycan deficiency and gamma-sarcoglycan deficiency. Acta Neuropathol (Berl) 1998;96:202–6. [31] Guglieri M, Magri F, D’Angelo MG, et al. Clinical, molecular, and protein correlations in a large sample of genetically diagnosed Italian limb girdle muscular dystrophy patients. Hum Mutat 2008;29:258–66. [32] Crosbie RH, Lim LE, Moore SA, et al. Molecular and genetic characterization of sarcospan: insights into sarcoglycan–sarcospan interactions. Hum Mol Genet 2000;9:2019–27. [33] Gouveia TL, Kossugue PM, Paim JF, et al. A new evidence for the maintenance of the sarcoglycan complex in muscle sarcolemma in spite of the primary absence of delta-SG protein. J Mol Med 2007;85:415–20.
L. Klinge et al. / Neuromuscular Disorders 18 (2008) 934–941 [34] Vainzof M, Moreira ES, Canovas M, et al. Partial alpha-sarcoglycan deficiency with retention of the dystrophin–glycoprotein complex in a LGMD2D family. Muscle Nerve 2000;23:984–8. [35] Duggan DJ, Fanin M, Pegoraro E, et al. alpha-Sarcoglycan (adhalin) deficiency: complete deficiency patients are 5% of childhood-onset dystrophin-normal muscular dystrophy and most partial deficiency patients do not have gene mutations. J Neurol Sci 1996;140:30–9. [36] Ginjaar HB, van der Kooi AJ, Ceelie H, et al. Sarcoglycanopathies in Dutch patients with autosomal recessive limb girdle muscular dystrophy. J Neurol 2000;247:524–9. [37] Barresi R, Confalonieri V, Lanfossi M, et al. Concomitant deficiency of beta- and gamma-sarcoglycans in 20 alpha-sarcoglycan (adhalin)-deficient patients:
[38]
[39]
[40] [41]
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immunohistochemical analysis and clinical aspects. Acta Neuropathol (Berl) 1997;94:28–35. Bonnemann CG, Passos-Bueno MR, McNally EM, et al. Genomic screening for beta-sarcoglycan gene mutations: missense mutations may cause severe limbgirdle muscular dystrophy type 2E (LGMD 2E). Hum Mol Genet 1996;5:1953–61. Duclos F, Broux O, Bourg N, et al. Beta-sarcoglycan: genomic analysis and identification of a novel missense mutation in the LGMD2E Amish isolate. Neuromuscul Disord 1998;8:30–8. Politano L, Nigro V, Passamano L, et al. Evaluation of cardiac and respiratory involvement in sarcoglycanopathies. Neuromuscul Disord 2001;11:178–85. Meena AK, Sreenivas D, Sundaram C, et al. Sarcoglycanopathies: a clinicopathological study. Neurol India 2007;55:117–21.
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Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Sarcoglycanopathies: Can muscle immunoanalysis predict the genotype? Lars Klinge a, Gabriele Dekomien b, Ahmed Aboumousa a, Richard Charlton a, Jörg T. Epplen b, Rita Barresi a, Kate Bushby a, Volker Straub a,* a b
Institute of Human Genetics, University of Newcastle upon Tyne, International Centre for Life, Central Parkway, NE1 3BZ Newcastle-upon-Tyne, UK Ruhr-University Bochum, Human Genetics, Universitätsstraße 150, 44801 Bochum, Germany
a r t i c l e
i n f o
Article history: Received 9 June 2008 Received in revised form 24 July 2008 Accepted 16 August 2008
Keywords: Muscular dystrophy Sarcoglycanopathy Sarcoglycans Dystroglycan Immunoanalysis
a b s t r a c t Muscle immunoanalysis of the sarcoglycan complex is an important part of the diagnostic evaluation of muscle biopsies in patients with autosomal recessive limb-girdle muscular dystrophy. Reduced or absent sarcolemmal expression of one or all of the four sarcoglycans (a-, b-, c-, d-sarcoglycan) can be found in patients with limb-girdle muscular dystrophy 2C-F (LGMD2C-F) and also in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). It has previously been suggested that different patterns of sarcoglycan expression could predict the primary genetic defect, and that genetic analysis could be directed by these patterns. In this first UK study we studied 24 genetically characterized patients with sarcoglycan deficient LGMD, in 22 of whom muscle immunoanalysis data were available. Thirteen patients showed asarcoglycan deficient LGMD2D, 7 patients b-sarcoglycan deficient LGMD2E, 3 patients c-sarcoglycan deficient LGDM2C, and one patient d-sarcoglycan deficient LGMD2F. Muscle biopsies were analysed in one centre without knowledge of the established genetic diagnosis. Our results demonstrated that residual sarcoglycan expression is highly variable and does not enable an accurate prediction of the genotype. Considering previous reports of sarcoglycanopathy patients with an isolated loss of one sarcoglycan we recommend to use antibodies against all four sarcoglycans for immunoanalysis of skeletal muscle sections. A concomitant reduction of dystrophin and b-dystroglycan was observed more frequently than previously reported and illustrates the important differential diagnosis of DMD and BMD for sarcoglycan deficient LGMD. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction The autosomal recessive limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of disorders leading to progressive muscle wasting and weakness, with evidence for at least 14 distinct loci at present (http://www.musclegenetable.org). Four of these (LGMD2C-F) are caused by mutations in the genes coding for the sarcoglycan proteins a-, b-, c-, and d-sarcoglycan (collectively also referred to as sarcoglycanopathies) [1–5]. Two further sarcoglycans have also been identified (e-, and f-sarcoglycan [6– 8]). c-, d-, and f-sarcoglycan share significant sequence similarity while a-sarcoglycan is highly related to e-sarcoglycan. Mutations in the e-sarcoglycan gene are associated with myoclonus dystonia syndrome in humans [9], while no disease has so far been associated with mutations in the f-sarcoglycan gene. In skeletal and cardiac muscle a-, b-, c-, and d-sarcoglycan assemble into a tetrameric complex forming a subcomplex within the dystrophin glycoprotein complex (DGC). The sarcoglycans are glycosylated proteins with single transmembrane domains [10], and correct assembly of the * Corresponding author. Tel.: +44 1912418655; fax: +44 1912418770. E-mail address: [email protected] (V. Straub). 0960-8966/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.08.003
sarcoglycan complex is required for maintenance of the sarcolemma [11–13]. Their function is not completely understood but they appear to have both mechanical and non-mechanical roles that mediate interactions between the extracellular matrix, the sarcolemma and the cytoskeleton [14,15]. b-, d-, e-, and f-sarcoglycan have also been found to be important for myelin stability in Schwann cells [16]. Mutations in the gene for a-sarcoglycan are generally the most common sarcoglycan mutations, whereas mutations in d-sarcoglycan are the rarest. Due to a founder mutation in the c-sarcoglycan gene LGMD2C is the most common sarcoglycanopathy in the North African population [17]. In order to reach a diagnosis and provide accurate counselling for patients affected by sarcoglycanopathy, it is necessary to identify which sarcoglycan protein is primarily affected. So far, this can only be achieved through mutation analysis. Muscle immunoanalysis with antibodies against a-, b-, c-, and d-sarcoglycan is part of the diagnostic work-up of patients with autosomal recessive LGMD. However, a deficiency of any single component of the sarcoglycan protein complex may lead to concomitant reduction or loss of the other sarcoglycans [10,11,18–20]. Therefore, it would be of great advantage to identify expression patterns that could help to develop a diagnostic algorithm for the consecutive molecular
L. Klinge et al. / Neuromuscular Disorders 18 (2008) 934–941
analysis of the four sarcoglycan genes. Some studies have been published showing distinct patterns of sarcoglycan expression/ labelling [10,11,18–20], but so far data are not entirely consistent necessitating further investigation [19]. In order to further explore the feasibility of a diagnostic algorithm to direct sarcoglycan gene mutation analysis, we analysed clinical, biochemical, and molecular findings in a cohort of 24 patients with sarcoglycanopathy from the UK. The main aim of this study was to investigate if muscle immunoanalysis could correctly predict which of the four sarcoglycan genes was primarily affected in these patients.
2. Materials and methods We present data of 24 patients with sarcoglycanopathy studied at the Newcastle muscle centre, UK. These patients originated from 20 different families (13 patients with a-sarcoglycanopathy from 10 families, 7 patients with b-sarcoglycanopathy from 6 families, 3 patients with c-sarcoglycanopathy from 3 families and one patient with d-sarcoglycanopathy). From this cohort muscle biopsies were available for 22 patients. The clinical history of the patients was reviewed, and muscle sections and Western blots were analysed and rated by three independent investigators, all experienced in the interpretation of muscle immunoanalysis. Biopsy material was not suitable/not interpretable for immunohistochemistry in 3 patients, and only Western blots were analysed. No Western blot was available in one patient.
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1:100 in 0.1 M lysine in 40% Foetal Calf Serum. Sections were washed, visualised with diaminobenzidine tetrahydrochloride (DAB) and counterstained with Carazzi’s haematoxylin prior to dehydration and mounting. Control sections were labelled without primary antibodies, and all sections were compared with samples with non-LGMD neuromuscular disorders, and with normal muscle. 2.3. Multiplex immunoanalysis on Western blots Multiplex immunoanalysis on Western blots from biphasic polyacrylamide gels (4–12% gradient) was performed using antibodies to dysferlin (NCL-hamlet exon 53), dystrophin (Dy8/6C5 C-terminus and Dy4/6D3 rod domain), a-sarcoglycan, b-dystroglycan, c-sarcoglycan, calpain 3 (NCL-CALP-2C4 and NCL-CALP-12A2), and caveolin 3 (BD610420). Myosin heavy chain staining on the post-blotted gel was used as loading control. 2.3.1. Evaluation of biopsies Three independent evaluators, all experienced in reading of muscle biopsies and Western blots, evaluated the biopsies and were blinded as to the underlying genotype of the patients. Protein expression on sections and blots was scored into normal +++, slight reduction ++, reduction +, severe reduction +/ , and absence . b-Spectrin was used as a marker of membrane integrity on immunohistochemistry. Based on the expression pattern of the sarcoglycans and other proteins of the DGC, the evaluators made a prediction of the underlying genotype of the patients.
2.1. Mutation analysis Screening for mutations was performed by polymerase chain reaction (PCR) for all exons of the four sarcoglycan (SGC) genes (SGCA, 9 exons; SGCB, 6 exons; SGCG, 8 exons; SGCD, 8 exons) by denaturating high performance liquid chromatography (DHPLC; transgenomic Cheshire, UK; analysis software Wavemaker 4.1). DNA samples with deviating melting curves were directly sequenced (MegaBace1000 Amersham). In order to exclude intragenic deletions multiplex ligation-dependent probe amplification (MLPA) was applied in all patients with homozygous mutations and in patients with only a single mutation detected by DHPLC analysis.
3. Results 3.1. Mutation analysis Mutation analysis showed the following distribution of sarcoglycan mutations either in homozygous or compound heterozygous status in our cohort: a-sarcoglycan 54%, b-sarcoglycan 29%, c-sarcoglycan 13%, and d-sarcoglycan 4%. Of these mutations 11 were novel. The mutations and the predicted molecular consequences are listed in Table 1. The 3 LGMD2C patients in our cohort all had homozygous missense mutations.
2.2. Muscle biopsy Immunoanalysis 3.2. Clinical information Muscle biopsies were analysed as part of the National Commissioning Group (NCG) designated specialised diagnostic service for Limb-Girdle Muscular Dystrophies (LGMD). Optimised immunohistochemical and multiplex Western blot protocols were used for the demonstration and resolution of muscular dystrophy associated proteins as previously described [21]. Open or needle biopsies of muscle were snap frozen in isopentane cooled in liquid nitrogen, mounted in OCT and 6lm sections were cut and mounted onto SuperFrostÒ Plus slides. Immunolabelling was carried out using a standard protocol. Briefly, sections were equilibrated to room temperature and washed for 15 min in PBS pH 7.3 containing 0.1% Triton X to permeabilise the membranes. Excess buffer was removed and sections incubated in optimally diluted primary antibody to b-spectrin (RBC2/3D5), b-dystroglycan (43DAG/8D5), C-terminus dystrophin (Dy8/6C5) and N-terminus dystrophin (Dy10/12B2), a-sarcoglycan (Ad1/20A6), b-sarcoglycan (1/5B1), c-sarcoglycan (35DAG/ 21B5), d-sarcoglycan (3/12C1) (all from Novocastra). All antibodies were diluted in 40% Foetal Calf Serum containing 0.1 M lysine and incubated overnight at 4 °C. Following 2 10 min washes in PBS/Triton sections were incubated for 90 min at room temperature in HRP conjugated rabbit anti mouse Ig (Dako P260) diluted
The clinical details of the patients are listed in Table 2. All patients showed limb-girdle weakness, calf hypertrophy and elevated serum creatine kinase levels (4–100 times the normal value). Age of onset varied from 1.5 up to 30 years. The phenotype was graded into mild, intermediate and severe depending on the age of onset and progression of the disease. Mean age of onset in a-sarcoglycanopathy was 13.2 ± 2.3 years, 6.5 ± 1.1 years in b-sarcoglycanopathy, and 6.7 ± 1.4 years in c-sarcoglycanopathy. Overall, the progression of symptoms in patients with a-sarcoglycanopathy was diverse: 4 patients lost ambulation before the age of 16 years while nine patients are still ambulant, 3 of them aged 50 years and above. Patients with b-sarcoglycanopathy appeared to display a slightly more consistent and severe phenotype with 4/7 patients having lost ambulation before the age of 25 years. Echocardiography data were available in 21 patients. The mean age at which echocardiography was performed was 22.3 years. None of the patients showed evidence of cardiomyopathy. Two patients (one affected by a-, and one by b-sarcoglycanopathy) needed nocturnal ventilator support from the age of 26 and 37 years on.
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L. Klinge et al. / Neuromuscular Disorders 18 (2008) 934–941
Table 1 Molecular data of the patients No.
Allele 1
Allele 2
Type
Mutation
Location of mutation allele 1
Location of mutation allele 2
1 2a
c.695G>C(p.Cys232Ser) c.347_351dupCTCGG (Gln118LeufsX94)
c.695TG>C(p.Cys232Ser) c.371C>T(p.Ile124Thr)
New
Extracellular Extracellular
Extracellular Extracellular
2b
c.347_351dupCTCGG (Gln118LeufsX94)
c.371C>T(p.Ile124Thr)
Extracellular
Extracellular
3
c.347_351dupCTCGG (Gln118LeufsX94)
c.229C>T(p.Arg77Cys)
Extracellular
Extracellular
4 5
c.292C>A (p.Arg98Ser) c.229C>T(p.Arg77Cys)
c.292C>A(p.Arg98Ser) c.739G>A(p.Val247Met)
Extracellular Extracellular
Extracellular Extracellular
6a
c.229C>T(p.Arg77Cys)
c.739G>A(p.Val247Met)
Extracellular
Extracellular
6b
c.229C>T(p.Arg77Cys)
c.739G>A(p.Val247Met)
Extracellular
Extracellular
7
c.371C>T (p.Ile124Thr)
c.739G>A(p.Val247Met)
Extracellular
Extracellular
8
c.613C>T(p.Pro205Ser)
c.265C>T(p.Leu89Phe)
Missense Frameshift/ missense Frameshift/ missense Frameshift/ missense Missense Missense/ missense Missense/ missense Missense/ missense Missense/ missense Missense
Extracellular
Extracellular
9a
c.371C>T (p.Ile124Thr)
c.850C>T (p.Arg284Cys)
Extracellular
Extracellular
9b
c.371C>T (p.Ile124Thr)
c.850C>T (p.Arg284Cys)
Extracellular
Extracellular
10
c.293C>A (p.Arg98His) and in c: SGC exon 8 c.778_779del AGSer360ProfsX57)
488insGLeu164ThrfsX27
New, allele 2
Extracellular
Extracellular
11
IVS2+5G>A
IVS2+5G>A
New
Intracellular
Intracellular
12a 12b 13
c.23_54 ins c.23_54 ins, exon 1 c.1_2delAT
c.23_54 ins c.23_54 ins c.341C>T(p.Ser114Phe)
New New New
Intracellular Intracellular Intracellular
Intracellular Intracellular Extracellular
14
exon 1 c.1_2delAT
c.341C>T(p.Ser114Phe)
New
Intracellular
Extracellular
15 16
c.341C>T(p.Ser114Phe) c.341C>T(Ser114Phe)
c.341C>T(p.Ser114Phe)
Extracellular Extracellular
Extracellular
LGMD2C (csarcoglycan)
17 18 19
c.244G>A (p.Gly82Arg) c.158T>C (p.Leu53Pro) c.158T>C (p.Leu53Pro)
c.244G>A (p.Gly82Arg) c.158T>C (p.Leu53Pro) c.158T>C (p.Leu53Pro)
Missense Missense Missense
New New New
Extracellular TM TM
Extracellular TM TM
LGMD2F (dsarcoglycan)
20
c.226G>T (p.Gly76Cys)
c.226G>T (p.Gly76Cys)
Missense
New
Extracellular
Extracellular
LGMD2D (asarcoglycan)
LGMD2E (bsarcoglycan)
Missense/ missense Missense/ missense Missense/ frameshift Splice mutation Duplication Duplication Frameshift/ missense Frameshift/ missense Missense Missense
New
Both new
No., patient number; TM, transmembrane.
Table 2 Clinical data of the patients No.
Age at onset (years)
Functional state
1 2a 2b 3 4 5 6a 6b 7 8 9a 9b 10
6 8 7 4 5 30 18 15 15 28 14 14 7
Lost ambulation Lost ambulation Lost ambulation Lost ambulation Climb stairs 6 Ambulant at 50 Ambulant at 56 Ambulant at 50 Ambulant at 26 Ambulant at 34 Ambulant at 40 Ambulant at 38 Climb stairs 10
11 12a 12b 13 14 15 16
6 7 10 8 1.5 9 4
LGMD2C
17 18 19
LGMD2F
20
LGMD2D
LGMD2E
Cardiac.
Resp.
Skel. deform.
CK
Age (years) at CK
n.a. No No No No No n.a. n.a. No No No No No
n.a. No No Yes No No n.a. n.a. No No No No No
n.a. No No Scoliosis No No n.a. n.a. No No No No No
15.77 5000 2500 High 2736 2000 1160 n.a. 5296 1900 6000 1700 12.297
7 14 16 n.a. 5 50 56
Lost ambulation at 11 Ambulant at 28 Ambulant at 25 Lost ambulation at 25 Lost ambulation at 15 Lost ambulation at 14 Occasional wheelchair at 11
No No No No No No No
No No No Yes No No No
No No No Scoliosis No Scoliosis No
800 2000 3358 8812 4640 7000 2300
3 8 9
With aid at 8y With aid at 30 Ambulant at 11
No No n.a.
No No n.a.
No Scoliosis n.a.
19.244 950 n.a.
7 21
5
Lost ambulation at 12
n.a.
n.a.
n.a.
19,000
12
at at at at
7 10 10 15
21 32 31 26 10 26 23 12 9 7
No., patient number, n.a., not available, yrs., years; Cardiac, cardiac involvement; resp., respiratory involvement, skel. deform., skeletal deformities; CK, serum Creatin Kinase.
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L. Klinge et al. / Neuromuscular Disorders 18 (2008) 934–941 Table 3 Muscle immunoanalysis data Immunohistochemistry
LGMD2D
LGMD2E
LGMD2C
LGMD2F
No.
Phenotype
a-SG
1 2a 3 4 5 6a 6b 7 8 9a 10
Severe Severe Severe Severe Mild Mild Mild Intermediate Mild Mild Severe
+
11 12a 12b 13 14 15 16
Severe Intermediate Intermediate Severe Severe Severe Severe
17 18 19
Severe Intermediate Too young to judge
20
Severe
c-SG
b-SG
d-SG
b-Spectrin
b-DG
Dys-N
Dys-C
a-SG
++ + + +/
++ + + +/
+ +++ +++ +++
+ ++ +++ +
++ +++ +++ ++
++ +++ +++ ++
+/
+
++
+
+++
++
++
+++
+ +/ +
++ + + +/
++ + + +/
+++ +++ +++ +++
+++ ++ +++ +
+++ ++ +++ ++
+++ +++ +++ ++
+/ +/ + ++ + + + +/
+/ +/ + +/-
+ +
+/ + + + + n.a. +/
++ +++ +++ ++ +++ ++ ++
+ + + +++ + + +/
+++ +++ ++
+++ +++ +++
+ +/
++ ++ +++
+++ +++ +++
+/ + ++
++ ++ +++
+/ ++ ++
+ + ++
++ +++ +++
++
+/
++
++
+/
Not suitable + Not suitable + + +
+ + Not suitable + +
+ +
Western Blot
+ n.a.
+ +/
+++, normal; ++, slightly reduced; +, reduced; +/ , severely reduced;
+/n.a.
c-SG
+/
+ +
-
+ + +/
b-DG
Dys-N
Dys-C
Predicted diagnosis
++ ++ ++ ++ + ++ ++
+++ +++ +++ +++ ++ +++ ++ +++ +++ +++ +++
+++ +++ +++ +++ ++ +++ ++ +++ +++ +++ +++
LGMD2C LGMD2D Impossible Impossible Impossible LGMD2C Impossible LGMD2C LGMD2C Impossible Impossible
+ +/
++ +++ ++
++ +++ +++
+
++
++
Impossible Impossible LGMD2C LGMD2C LGMD2C
++ +++ ++
+/
+
+
LGMD2D
++ ++ ++
+ ++ +++
++ +++ +++
LGMD2C LGMD2C LGMD2C
+
++
++
LGMD2D
, absent; No., patient number; SG, sarcoglycan; b-DG, b-dystroglycan; Dys, dystrophin.
3.3. Correlation of residual sarcoglycan expression, mutation and clinical phenotype Nine patients with a-sarcoglycanopathy showing an intermediate or mild phenotype retained some residual expression of a-sarcoglycan. In those patients displaying a severe phenotype 4 out of 5 showed complete absence of a-sarcoglycan indicating a possible relation of clinical severity and residual expression of a-sarcoglycan in LGMD2D. All patients with residual staining for a-sarcoglycan had missense mutations. This is in accordance with the observation that missense mutations seem to be associated with a milder phenotype while truncating mutations tend to result in a more severe course [22,23]. In b-sarcoglycanopathy 2 patients showed a severe phenotype, 2 of whom had complete loss of b-sarcoglycan staining. One of these patients had a missense and the other one a splice site mutation. The patient with a severe phenotype and residual b-sarcoglycan expression was compound heterozygous with a frameshift mutation on one and a missense mutation on the other allele. In contrast to the patients with LGMD2D all of the LGMD2C patients in our cohort had missense mutations and complete absence of c-sarcoglycan on sections. In the patient with d-sarcoglycanopathy a missense mutation led to a complete absence of the sarcoglycan complex on sections. 3.4. Muscle immunoanalysis (Table 3) The sarcoglycans analysed by Multiplex Western blot were a-sarcoglycan (50 kDa) and c-sarcoglycan (35 kDa). Due to overlap or proximity in molecular size analysis of b-sarcoglycan (43 kDa) and d-sarcoglycan (35 kDa) was not possible within the multiplex method. 3.5. LGMD2D 3.5.1. Immunohistochemistry In LGMD2D patients a-sarcoglycan was absent in 4 out of 9 patients and the most severely reduced protein in one patient. Only
in this 1 patient a correct prediction of the primary defect was made. In 4 patients, c-sarcoglycan was completely absent. In 2 patients, c-sarcoglycan was more severely reduced than a-sarcoglycan. In all LGMD2D patients, it could be noted that b- and dsarcoglycan showed a reduced but preserved expression. b-Dystroglycan was reduced in six patients, while dystrophin was reduced in 5 patients. 3.5.2. Western blot analysis a-Sarcoglycan was absent in 2 out of 11 patients, severely reduced in 4 patients, slightly reduced in one patient. The expression of c-sarcoglycan was affected in all patients: absent in 8, severely reduced in one, and reduced in 2 patients. In 8 patients, the reduction of c-sarcoglycan was more pronounced than the one of the primarily involved a-sarcoglycan. b-Dystroglycan was similarly reduced in all but one case, whereas dystrophin was reduced in 2 patients. 3.6. LGMD2E 3.6.1. Immunohistochemistry In LGMD2E patients b-sarcoglycan was absent in 2 patients. Here a- and c-sarcoglycan were also completely absent and dsarcoglycan was partially preserved. In those 3 patients with low residual expression of b-sarcoglycan c-sarcoglycan was found to be more severely reduced than b-sarcoglycan, and d-sarcoglycan showed a similar expression as b-sarcoglycan. b-Dystroglycan was overall more severely reduced than in a- or c-sarcoglycanopathy, and dystrophin was slightly reduced in three cases. 3.6.2. Western Blot analysis In LGMD2E patients a-sarcoglycan was absent in 2, severely reduced in 2 and reduced in 2 patients. c-Sarcoglycan was absent in all patients. b-Dystroglycan was absent in 1 patient, severely reduced in 2 patients, and reduced in 3 patients. Dystrophin was affected in all but 1 patient.
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3.7. LGMD2C 3.7.1. Immunohistochemistry c-Sarcoglycan was the protein most often and most severely reduced across the whole spectrum of sarcoglycanopathies. In LGMD2C patients, c-sarcoglycan was absent in 3 of 3 patients. In 2 patients, there was partial preservation of the other sarcoglycans. In 1 patient with c-sarcoglycanopathy also a-, and b-sarcoglycan were completely absent with partial preservation of d-sarcoglycan. b-Dystroglycan and dystrophin expression were compromised in all 3 patients. 3.7.2. Western Blot In LGMD2C patients, a-sarcoglycan was reduced in 2 and absent in the third patient, whereas c-sarcoglycan was absent in all patients. b-Dystroglycan was affected in all patients, and dystrophin was affected in 2 patients.
can was also reduced, and reduction of the N-terminal dystrophin domain was more pronounced than that of the C-terminal domain. Overall, Western blot results showed clear reduction of the sarcoglycans and of other DGC components, but the high variability within this pattern did not allow prediction of the primary sarcoglycan gene involved. The sensitivity of the antibody against c-sarcoglycan is slightly lower on Western blots than the antibody against a-sarcoglycan which could contribute to the fact that csarcoglycan is more often and more pronounced reduced on Western blot. This characteristic may be helpful in detecting subtle changes in the expression pattern of c-sarcoglycan. A correct prediction of the mutated gene by the investigators who analysed the biopsy samples was possible in 19%, the prediction was ambiguous in 38%, and wrong in 43% of our samples. Therefore muscle immunoanalysis did not accurately predict the primary defect in the majority of sarcoglycanopathy patients. 4. Discussion
3.8. LGMD2F 3.8.1. Immunohistochemistry Our data on d-sarcoglycanopathy are based on observations in 1 patient and therefore of limited significance. In this patient all sarcoglycans were absent, b-dystroglycan was severely reduced and dystrophin slightly reduced. 3.8.2. Western Blot Absence of a-sarcoglycan, a severe reduction of c-sarcoglycan, a reduction of b-dystroglycan and slight reduction of dystrophin were observed in this patient. 3.9. Prediction of sarcoglycan primarily involved, based on immunohistochemistry and Western blot In 100% of cases, all of the compounds of the sarcoglycan complex were reduced on immunohistochemistry, but our data did not allow the identification of specific expression patterns relating to the primary gene involved, see Table 3. On Western blotting aand c-sarcoglycan were reduced in expression across all four sarcoglycanopathies. The majority of samples showed an additional reduction in the expression of other DGC proteins, especially of b-dystroglycan (16/ 19 cases on immunohistochemistry and 20/21 on Western blot). This reduction was less pronounced in LGMD2D (6/9 cases on immunohistochemistry) and most apparent in LGMD2E patients. In accordance with the biochemical structure of the DGC, dystrophin only showed a reduced expression pattern when b-dystrogly-
We present biochemical data on 22 and clinical and genetic data on 24 patients in the first UK study on genetically proven sarcoglycanopathy. In these patients, we were able to identify 11 novel mutations (7 missense, 2 frame shift, 1 splice site mutation and one duplication, Table 1). Comprehensive muscle immunoanalysis in our cohort of patients unequivocally showed a reduction of all four sarcoglycans and other proteins of the DGC, but no specific expression patterns did emerge which would help to establish an algorithm for mutation analysis (Table 3). Specific patterns of abnormal sarcoglycan expression have on the other hand previously been described in sarcoglycanopathies, especially in LGMD2C. It was suggested that patients with LGMD2C show a significantly reduced or absent staining of c-sarcoglycan with reduced but partly preserved staining of the other sarcoglycan proteins, in particular a- and d-sarcoglycan [12,19,20,22,24– 31]. We found a similar pattern in our patients with c-sarcoglycanopathy, but intriguingly this pattern was not specific for LGMD2C as it was also found in some of our patients with a- and b-sarcoglycanopathy and contributed to the fact that 7 of these patients were misdiagnosed as LGMD2C. Therefore, this staining pattern may be more variable and unspecific across the different forms of sarcoglycanopathy than previously anticipated, especially in those cases with some residual expression of c-sarcoglycan (Table 3). The inconsistent staining pattern in LGMD2C is also illustrated by a patient who showed normal expression of a-, b-, and d-sarcoglycan [32]. Complete maintenance of the other sarcoglycans despite the loss of d-sarcoglycan has also been shown in a patient with LGMD2F [33].
Fig. 1. Different patterns of muscle immunohistochemistry findings in 2 patients affected by a-sarcoglycanopathy (both with missense mutations in the extracellular domain). The insets represent labelling of control muscle. Patient 6a shows a more severe phenotype than patient no. 4. Magnification 20.
L. Klinge et al. / Neuromuscular Disorders 18 (2008) 934–941
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Fig. 2. Muscle immunoanalysis in a patient with Becker muscular dystrophy due to a duplication in the dystrophin gene: immunohistochemistry shows normal dystrophin labelling (antibody directed against the C-terminus) and a reduction of a-sarcoglycan and b-dystroglycan staining. Western blot reveals the increase in molecular weight of dystrophin and, in contrast to immunohistochemistry, demonstrates normal expression of a-sarcoglycan and c-sarcoglycan. Magnification 10.
While some studies have found a-sarcoglycan to be most severely reduced in primary a-sarcoglycanopathy [20,23,24, 30,34,35], other investigations demonstrated a more variable expression pattern from total absence of the entire sarcoglycan complex [22], to some preservation of c-sarcoglycan only [22,36] and to a similar reduction of a- and b-sarcoglycan [36]. In contrast, another study found that severe reduction of a- and b-sarcoglycan did not occur together at all [37]. This inconsistent pattern of sarco-
glycan staining was also evident in our study (Fig. 1). In our cohort of 11 LGMD2D patients, we were able to correctly predict the genotype in only one patient in whom a-sarcoglycan was most reduced on sections and absent on Western blotting. In 6 patients, it was impossible to predict the genotype because c-sarcoglycan was similarly reduced or absent on sections and blots, while b- and d-sarcoglycan were relatively well preserved. In these 6 patients bdystroglycan was best preserved, suggesting and important role
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for b- and d-sarcoglycan to maintain integrity of the DGC. No correlation was found between residual expression of a-sarcoglycan and the site of mutation. Data on the immunoanalysis profile in b-sarcoglycanopathy are also not entirely consistent. Three studies have found the whole sarcoglycan complex to be absent in LGMD2E [22,24,38], one study found near absence or absence of all four sarcoglycans [20], and one study found some residual expression of c-sarcoglycan only [36] (27 patients in total). In contrast, in our group of 6 LGMD2E patients we found residual expression of some of the sarcoglycans in all of them but were unable to predict the genotype correctly in any of them. This was mainly attributed to the fact that a- and c-sarcoglycan were similarly or more reduced than b-sarcoglycan leading to a wrong prediction in four, and making a prediction impossible in two cases. In LGMD2E b-dystroglycan was more markedly reduced than in LGMD2D, again suggesting a closer association between bsarcoglycan and other components of the DGC. Reduction of b-dystroglycan on immunofluorescence has previously been reported in 63% of patients with sarcoglycanopathy [22]. There has also been a report of a reduction of a-dystroglycan in a patient with LGMD2E [39]. We found b-dystroglycan to be reduced in 84% of our patients on immunohistochemistry and in 95% on Western blot. Dystrophinopathy is an important differential diagnosis in patients with sarcoglycanopathy. Immunohistochemical analysis of muscle sections alone can be misleading, as depicted in Fig. 2 in a patient with a duplication in the dystrophin gene. In contrast to immunohistochemistry, which showed normal dystrophin and patchy sarcoglycan and b-dystroglycan staining, Western blot analysis revealed normal expression of a- and c-sarcoglycan and an altered band for dystrophin. In addition, labelling of sections with nNOS and utrophin may provide useful information in some cases as well. Cardiac involvement is a known complication in patients with sarcoglycanopathy. In a study by Politano et al. 1 of 3 LGMD2D patients, 2 of 2 LGMD2E patients, 6 of 9 LGMD2C patients, and 2 of 2 LGMD2F patients had evidence of heart involvement. Seven of these 11 affected patients were presymptomatic [40]. In contrast, cardiac findings in our patients are similar to a recent study on 24 sarcoglycanopathy patients in whom cardiac abnormalities were only found in 1 patient [41]. This suggests that cardiac involvement in sarcoglycanopathy may be less common than previously anticipated, even in LGMD2F. Although predictive patterns of residual sarcoglycan expression may be present in some patients, we conclude that generally sarcoglycan expression may vary significantly in patients with sarcoglycanopathy. Therefore, we recommend labelling of patient biopsies with all four sarcoglycan antibodies in order not to miss a possible isolated loss of any one of the sarcoglycans, as can be the case in some patients [32,33]. As muscle immunoanalysis cannot accurately predict the genotype, genetic analysis should be guided by the known prevalence patterns for LGMD2C, 2D, 2E, and 2F. Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft (KL 1868/1-1 to L.K.), G.D., V.S., H.L., J.T.E., and L.K. are members of the German Muscular Dystrophy Network (MD-NET 01GM0601) funded by the German Ministry of Education and Research (BMBF, Bonn, Germany); http://www.md-net.org. MD-NET is a partner of TREAT-NMD (EC, 6th FP, proposal # 036825; http://www.treat-nmd.eu). We are thankful to Louise Anderson for all her contributions to this study. References [1] Bonnemann CG, Modi R, Noguchi S, et al. Beta-sarcoglycan (A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex. Nat Genet 1995;11:266–73.
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Clinical, molecular, and protein correlations in a large sample of genetically diagnosed Italian limb girdle muscular dystrophy patients. Hum Mutat 2008;29:258–66. [32] Crosbie RH, Lim LE, Moore SA, et al. Molecular and genetic characterization of sarcospan: insights into sarcoglycan–sarcospan interactions. Hum Mol Genet 2000;9:2019–27. [33] Gouveia TL, Kossugue PM, Paim JF, et al. A new evidence for the maintenance of the sarcoglycan complex in muscle sarcolemma in spite of the primary absence of delta-SG protein. J Mol Med 2007;85:415–20.
L. Klinge et al. / Neuromuscular Disorders 18 (2008) 934–941 [34] Vainzof M, Moreira ES, Canovas M, et al. Partial alpha-sarcoglycan deficiency with retention of the dystrophin–glycoprotein complex in a LGMD2D family. Muscle Nerve 2000;23:984–8. [35] Duggan DJ, Fanin M, Pegoraro E, et al. alpha-Sarcoglycan (adhalin) deficiency: complete deficiency patients are 5% of childhood-onset dystrophin-normal muscular dystrophy and most partial deficiency patients do not have gene mutations. J Neurol Sci 1996;140:30–9. [36] Ginjaar HB, van der Kooi AJ, Ceelie H, et al. Sarcoglycanopathies in Dutch patients with autosomal recessive limb girdle muscular dystrophy. J Neurol 2000;247:524–9. [37] Barresi R, Confalonieri V, Lanfossi M, et al. Concomitant deficiency of beta- and gamma-sarcoglycans in 20 alpha-sarcoglycan (adhalin)-deficient patients:
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