Diagnosis of limb-girdle muscular dystrophy 2A by immunohistochemical techniques

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1 Neuropathology 2008; 28, doi: /j x Original Article Diagnosis of limb-girdle muscular dystrophy 2A by immunohistochemical techniques Hanna K. Kolski, 1 Cynthia Hawkins, 2 Mayana Zatz, 3 Flavia de Paula, 3 Doug Biggar, 4 Ben Alman 2 and Jiri Vajsar 2 1 Department of Pediatrics, University of Alberta, Edmonton, Alberta, 2 The Hospital for Sick Children, Toronto, and 4 Bloorview-MacMillan Center, Toronto, Ontario, Canada, and 3 Human Genome Research Center, Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil The Western blot technique is currently the standard detection method for suspected limb girdle muscular dystrophy (LGMD) 2A (calpainopathy). This is the first report in the English literature of the successful application of immunohistochemical techniques to support a diagnosis of LGMD 2A. This approach is straightforward and appears to be reasonably specific. We propose that immunohistochemical methods should be re-evaluated for the screening of undiagnosed patients with suspected LGMD 2A. Key words: calpain-3, immunohistochemistry, limb girdle muscular dystrophy 2A, myopathy, Western blot. INTRODUCTION Limb girdle muscular dystrophy (LGMD) 2A is caused by mutations in the calpain-3 (CAPN3) gene (also called p94 gene), which encodes CAPN3, the large skeletal-musclespecific member of the calpain family. 1 It has been reported to be the most frequent autosomal recessive form of muscular dystrophy in many populations. 2 6 Most limb girdle muscular dystrophies are screened using immunohistochemical techniques, including the sarcoglycanopathies and merosin-deficient muscular dystrophies. However, the Western blot technique is currently the accepted detection method for suspected LGMD 2A (calpainopathy). This, at least in part, derives from the perception that calpain-3 antibodies do not work on immunochemistry. 7,8 Correspondence: Hanna K. Kolski, md, Division of Pediatric Neurology, Section of Pediatric Neurosciences, Department of Pediatrics, 7317A, Aberhart Center #1, University Avenue, Edmonton, AB T6G 2J3, Canada. hanna.kolski@capitalhealth.ca Received 23 July 2007; revised 26 August 2007; accepted 5 October To the best of our knowledge, this manuscript is the first description in the English literature demonstrating that LGMD 2A can be effectively screened using immunohistochemical staining with commercially available calpain antibodies. There is one previous case report written in Japanese with successful application of immunostaining with a different monoclonal antibody against human calpain-3. 9 METHODS We systematically identified a cohort of 21 patients with proximal limb-girdle weakness and dystrophic changes on muscle biopsy. Appropriate ethical approval and informed consent were obtained, in accordance with the Declaration of Helsinki. Total protein was extracted from available muscle samples. Cell lysis was performed at boiling temperature using 20 mm Tris-HCl, ph 7.4, containing 2% SDS, 5 mm ethyleneglycoltetracetic acid (EGTA), 5 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride (PMSF), 5 mg leupeptin/ml and 10 mg aprotinin/ml. Measured aliquots of the extracted proteins were subjected to SDS-PAGE (7.5% acrylamide). Western-blot was performed using calpain-3 monoclonal antibodies calp3d/12a2 and calp3d/ 2C4 (Novocastra Laboratories (NCL), Newcastle-upon- Tyne, UK), and the reaction was detected by using horseradish peroxidase-conjugated anti-igg (1 U/mL; Boehringer) and staining with diaminobenzidine. The dilutions for NCL-CALP-2C4 were 1:25 1:50 and for NCL- CALP-12A2, 1:100. The muscle cryosections of all limb-girdle patients were then subjected to immunohistochemical staining as follows: 7-mm frozen tissue sections were mounted on positively charged microscope slides, air dried, and fixed in acetone for 10 min at room temperature. The sections were

2 Immunohistochemistry and calpain then incubated at room temperature with a 1:50 dilution of mouse antihuman calpain-3, clone 12A2 (Novocastra) for 60 min. Subsequent immunodetection was done using the Vector Elite ABC detection system (Vector Laboratories, Burlingame, CA, USA) according to manufacturer s instructions. Sections were counterstained with hematoxylin for nuclear detail. Appropriate positive controls (normal muscle) and negative controls were also performed in parallel. Mutation screening of the CAPN3 gene was performed using the single strand conformation polymorphism (SSCP) technique for confirmation in suspected cases. DNA samples were amplified by PCR. Upon screening of the calpain-3 gene through SSCP, samples with abnormal migration pattern on SSCP gel were sequenced in an automatic sequencer machine (ABI Model 377 Version 3.0). RESULTS A B Fig. 1 A and B: Western blots of muscle biopsies from patients with various limb girdle muscular dystrophies (LGMD). Approximate molecular masses (kda) are shown on the left. Both the 2C4 (A) and 12A2 (B) antibodies recognize full-size calpain-3 at 94 kd plus a number of smaller fragments, particularly at 60 and 30 kd. Note the absence/marked paucity of staining in lanes 2, 9, 10, suggestive of calpainopathy. The patient with the heterozygous 550delA mutation is represented in lane 10. Lane 4 corresponds to a muscle biopsy from a patient with alpha sarcoglycanopathy. The remaining samples are currently uncharacterized LGMD. Lanes 1 and 13 are positive and negative controls, respectively. We identified three patients with progressive predominantly proximal limb-girdle weakness and dystrophic muscle biopsies. In these cases, Western blots demonstrated an absence/marked paucity of antibody staining with the calpain 2C4 (exon 1) and 12A2 (exon 8) antibodies (Fig. 1A,B, respectively). This involved the 94 kda band, as well as the 60 and 30 kda bands. All patients had clinical findings consistent with LGMD 2A as previously described. 1,10,11 There was concordant absence of immunohistochemical 12A2 calpain antibody staining in all three of these muscle biopsy specimens (Fig. 2). Of note, calpain-3 was immunopositive in control muscles, as well as in other types of limb-girdle muscular dystrophies. In one patient, SSCP and screening of all coding regions of the calpain-3 gene revealed a heterozygous 550delA mutation (exon 4) but the second mutation was not identified, even after direct gene sequencing. In a second patient, DNA samples could not be obtained, while in the third one the DNA quality did not allow its amplification. The patient who had concordant laboratory evidence of calpainopathy presented with acquired progressive ambulatory difficulties at 6 years of age. This included frequent falls and problems with climbing stairs. At age 12, he was ambulant but relied on using the rails with stairs. On examination, he demonstrated scapular winging, decreased bulk in the upper arms, calf hypertrophy, and mild asymmetrical heel cord contractures. Gower s sign was absent. However, by MRC (Medical Research Council) scale measurements, he had hip- more than shoulder-girdle weakness (4/5), disproportionately weak hip extensors (2/5), and mild neck flexor weakness. Deep tendon reflexes were absent in upper extremities, but normal in lower extremities. Creatine phosphokinase ranged from 7744 to U/L. There were mild dystrophic changes in the right triceps muscle biopsy. By age 17 years he had progressed, with a full Gower s sign. Quadriceps were 2/5 and 2+/5 (right and left, respectively). Hip extensors were 2/5 and hamstrings 3/5. Pulmonary function tests were normal. Cardiac evaluation was normal including echocardiogram. His Western blot demonstrated absent staining of the

3 266 HK Kolski et al. A B Fig. 2 (A) Immunohistochemical staining with calpain-3 12A antibody of positive control muscle. (B) Immunohistochemical staining with calpain-3 12A antibody of LGMD 2A muscle biopsy. This is derived from the same specimen represented in lane 10 of Fig. 1A,B. There is lack of immunoreactivity. Magnification kda band with the calpain 2C4 antibodies and faint staining with the 12A2 antibodies (Fig. 1A,B lane 10). Immunohistochemical staining with the 12A2 antibody was absent (Fig. 2B). DISCUSSION To the best of our knowledge, this is the first report in the English literature of the successful application of immunohistochemical techniques to support a diagnosis of LGMD 2A and first report ever using calp3d/12a2 antibodies. One of our patients had convincing evidence of LGMD 2A, including a characteristic phenotype, and concordant diagnostic support from Western blot, immunohistochemistry, and mutational analysis. There is one previous case report written in Japanese with successful application of immunostaining with monoclonal antibodies against human calpain-3 (2C4). 9 This related to a 45-year-old woman who presented with progressive limb-girdle weakness from age 25 years. In this patient s muscle biopsy, calpain-3 was completely absent in the sarcoplasm with the ABC method of immunohistological staining. However, granular and striated patterns were noted in the control muscle. Diagnosis was confirmed by the absence of the 94 kda band in Western blot and a homozygotic mutation (Leu 189 Val) in the calpain gene. However, this group relied on the 2C4 antibody for staining.we are the first to demonstrate that the 12A2 antibody could be used reliably as well. In recent years, Keira et al. used their own rabbitderived polyclonal antibody to demonstrate the calpain-3 localization in myofibrils and myonuclei in vivo. 12 The commercially available antibodies substantiated the findings in Western blot but not on the human cryosections. Another group utilized immunofluorescence microscopy with rabbit polyclonal antibodies to identify the myofibril compartments where calpain-1 is concentrated. 13 However, the substrate tissue here was postmortem bovine skeletal red muscles. Thus, immunobased techniques have the advantage of facilitating calpain localization within cells. In contrast, it has been generally accepted that Western blot is the most appropriate method to screen for LGMD 2A. In fact, it has been cited that the limitation for the widespread use of immunodiagnosis for calpain-3 is that the current 2C4 and 12A2 antibodies only work on Western blots and not on tissue sections. 7,14 Nevertheless, Western blot has a number of technical limitations. For instance, in the case of calpain reduction, variability in timing of muscle handling and processing may subject the tissue to autolysis and protein degradation. 15,16 This would not be expected to be so much of an issue with immunohistochemistry, though so far such studies have not been completed. On a practical clinical basis, not all pathology labs have access to Western blotting. In those centers already using immunohistochemical staining for screening of the limbgirdle muscular dystrophies, calpain-3 could be readily added to the existing panel of antibodies. Furthermore, conventional immunoblot protocols require the solubilization of a significant portion of a muscle biopsy specimen (20 30 mg). 17 Some institutions may not have sufficient muscle tissue left apart from what is already mounted on the slides and frozen. Immunohistochemistry would be a feasible alternative to screen for calpain-3 deficiency under these circumstances. Diagnosing calpainopathy on the basis of Western blot, compared with mutational gene analysis, is not sensitive

4 Immunohistochemistry and calpain (52.5%) nor entirely specific (87.8%). 11 Many studies have shown that there is no direct correlation between the amount of calpain-3 detected on Western blots and the severity of the phenotype. 1,7,8 A normal amount of CAPN3 on Western blotting may be found in genetically proven calpainopathies, whereas calpain may be reduced in amount or absent in other forms of LGMD as a secondary effect. 1,5 At this point in time, it is not known how immunohistochemical staining would be affected in secondary reductions of calpain, as seen in dysferlinopathy, 11,18 fukutin-related protein limb-girdle dystrophy 1 and titinopathy. 19 This would warrant further study, including confirmation by mutation analysis. In one patient we found the 550 del A mutation in one allele. The veracity of the finding is supported by the 550 del A mutation being a frequent one in calpainopathy. 5,11 Furthermore, the detection of only one mutation is relatively common, occurring in about 10 43% of patients with LGMD 2A. 1,8,11 This 550delA mutation, at the exon 4, is a frameshift mutation which results in a premature stop codon. If homozygous, a total loss of protein would be expected on Western blot. In heterozygous cases, the final effect on the protein cannot be reliably predicted. This again underscores the importance of other techniques, such as immunohistochemistry, reported here, in confirming the definite diagnosis in patients where the mutation in the second allele cannot be found. The identification of a protein defect on muscle biopsy is extremely important to select candidate patients for subsequent mutation detection analysis. Mutation screening of the CAPN3 gene is labor-intensive, expensive and timeconsuming. 1 We propose that immunohistochemical techniques with commercial calpain antibodies offer a feasible, straightforward and reasonably specific screening alternative to Western blot analysis. ACKNOWLEDGMENTS This work was primarily funded by the Hospital for Sick Children Foundation. We also wish to thank Dr Ayako Ochi for translating the Japanese paper published in Rinsho Shinkeigaku. REFERENCES 1. Zatz M, Starling A. Calpains and disease. N Engl J Med 2005; 9: Chae J, Minami N, Jin Y et al. Calpain 3 gene mutations: genetic and clinico-pathologic findings in limbgirdle muscular dystrophy. Neuromuscul Disord 2001; 11: de Paula F, Vainzof M, Passos-Bueno MR et al. Clinical variability in calpainopathy: what makes the difference? Eur J Hum Genet 2002; 10: Dincer P, Leturcq F, Richard I et al. A biochemical, genetic, and clinical survey of autosomal recessive limb girdle muscular dystrophies in Turkey. Ann Neurol 1997; 42: Fanin M, Nascimbeni AC, Fulizio L, Angelini C. The frequency of limb girdle muscular dystrophy 2A in northeastern Italy. Neuromuscul Disord 2005; 15: Topaloglu H, Dincer P, Richard I et al. Calpain-3 deficiency causes a mild muscular dystrophy in childhood. Neuropediatrics 1997; 28: Anderson LV, Davison K, Moss JA et al. Characterization of monoclonal antibodies to calpain 3 and protein expression in muscle from patients with limb-girdle muscular dystrophy type 2A. Am J Pathol 1998; 153: Fanin M, Fulizio L, Nascimbeni AC et al. Molecular diagnosis in LGMD2A: mutation analysis or protein testing? Hum Mutat 2004; 24: Ibi T, Jing L, Nakao N, Minami N, Sahashi K. A case of LGMD2A identified with both western blot analysis and immunostaining of calpain 3 in biopsied muscle (in Japanese with English abstract). Rinsho Shinkeigaku 2000; 40: Beckmann JS, Bushby KM. Advances in the molecular genetics of the limb-girdle type of autosomal recessive progressive muscular dystrophy. Curr Opin Neurol 1996; 9: Saenz A, Leturcq F, Cobo AM et al. LGMD2A: genotype-phenotype correlations based on a large mutational survey on the calpain 3 gene. Brain 2005; 128: Keira Y, Noguchi S, Minami N, Hayashi YK, Nishino I. Localization of calpain 3 in human skeletal muscle and its alteration in limb-girdle muscular dystrophy 2A muscle. J Biochem (Tokyo) 2003; 133: Raynaud F, Fernandez E, Coulis G et al. Calpain 1 titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril. FEBS J 2005; 272: Fanin M, Pegoraro E, Matsuda-Asada C, Brown RH Jr, Angelini C. Calpain-3 and dysferlin protein screening in patients with limb-girdle dystrophy and myopathy. Neurology 2001; 56: Fanin M, Nascimbeni AC, Fulizio L, Trevisan CP, Meznaric-Petrusa M, Angelini C. Loss of calpain-3 autocatalytic activity in LGMD2A patients with normal protein expression. Am J Pathol 2003; 163:

5 268 HK Kolski et al. 16. Pogue R, Anderson LV, Pyle A et al. Strategy for mutation analysis in the autosomal recessive limb-girdle muscular dystrophies. Neuromuscul Disord 2001; 11: Anderson LV, Davison K. Multiplex Western blotting system for the analysis of muscular dystrophy proteins. Am J Pathol 1999; 154: Vainzof M, Anderson LV, McNally EM et al. Dysferlin protein analysis in limb-girdle muscular dystrophies. J Mol Neurosci 2001; 17: Hackman P, Vihola A, Haravuori H et al. Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin. Am J Hum Genet 2002; 71: