Diagnostic Value of Dystrophin Immunostaining in the Diagnosis of Duchenne and Becker Muscular Dystrophy Patients
DOI:
https://doi.org/10.3889/oamjms.2021.7612Keywords:
Duchenne/Becker muscular dystrophy, Immunohistochemical staining, Dystrophin, Multiplex ligation-dependent probe amplificationAbstract
Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive muscular disorders caused by the absence or reduction of the muscle cytoskeletal protein dystrophin. Standard procedures to detect deletion and duplication of the DMD gene use Multiplex Ligation-Dependent Probe Amplification (MLPA). However, genetic testing, such as MLPA, is not covered by the national insurance scheme in Indonesia. Immunohistochemical (IHC) staining of dystrophin from muscle biopsy in the form of Formalin-Fixed Paraffin-Embedded (FFPE) specimens can be an alternative method to detect dystrophin expression in protein levels to establish the diagnosis of DMD or BMD.
Objectives: To determinate sensitivity, specificity and accuracy of IHC analysis of dystrophin in DMD/BMD patient in comparison with the standard genetic testing, MLPA.
Methods: Twenty-six patients enrolled in this study were clinically diagnosed as DMD/BMD in Dr. Sardjito Hospital and Universitas Gadjah Mada Academic Hospital. Genomic DNA was isolated from 3 mL of EDTA-peripheral whole blood samples. The deletion and duplication of DMD genes were detected by MLPA. IHC examination was performed using a specific antibody dystrophin (DYS2). Complete loss of dystrophin staining indicated DMD, while partial loss of dystrophin staining indicated BMD. MLPA result was used as the gold standard to determine sensitivity, specificity, and accuracy of IHC technique using a 2x2 table.
Results: MLPA results revealed 18 (18/26; 69.3%) patients with deletion and 3 (3/26; 11.5%) patients with duplication. Five (5/26; 19.2%) patients who showed no deletion nor duplication were excluded from the analysis. Among 21 patients with deletion or duplication, 18 (18/21; 85.7%) patients were out-of-frame (DMD) and 3 (3/21; 14.3%) patients were in-frame (BMD). Six patients showed a discrepancy between the IHC and MLPA results with 9.5% (2/21) false positive and 19% (4/21) false negative. The sensitivity of dystrophin IHC was 77.78%, specificity 33.33%, positive predictive value 87.5%, negative predictive value 20%, and accuracy 71.43%.
Conclusion: Muscle biopsy followed by IHC can be one of the diagnostic tools to diagnose BMD or DMD, with high sensitivity. The protein-based strategy is probably the most efficient way to approach the diagnosis of Duchenne and Becker muscular dystrophy in limited health care settings.
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References
Cohn RD, Campbell KP. Molecular basis of muscular dystrophies. Muscle Nerve. 2000;23(10):1456-71. https://doi.org/10.1002/1097-4598(200010)23:10<1456:AID-MUS2>3.0.CO;2-T PMid:11003781 DOI: https://doi.org/10.1002/1097-4598(200010)23:10<1456::AID-MUS2>3.0.CO;2-T
Van Putten M, Hulsker M, Nadarajah VD, van Heiningen SH, van Huizen E, van Iterson M, et al. The effects of low levels of dystrophin on mouse muscle function and pathology. PLoS One. 2012;7(2):e31937. https://doi.org/10.1371/journal.pone.0031937 PMid:22359642 DOI: https://doi.org/10.1371/journal.pone.0031937
Malik V, Rodino Klapac LR, Viollet L, Mendell JR. Aminoglycoside-induced mutation suppression (stop codon readthrough) as a therapeutic strategy for Duchenne muscular dystrophy. Ther Adv Neurol Disord. 2010;3(6):379-89. https://doi.org/10.1177/1756285610388693 PMid:21179598 DOI: https://doi.org/10.1177/1756285610388693
Bladen CL, Salgado D, Monges S, Foncuberta ME, Kekou K, Kosma K, et al. The TREAT-NMD DMD global database: Analysis of more than 7,000 duchenne muscular dystrophy mutations. Hum Mutat. 2015;36(4):395-402. https://doi.org/10.1002/humu.22758 PMid:25604253 DOI: https://doi.org/10.1002/humu.22758
Nadifi S, Bellayou H, Hamzi K, Rafai MA, Karkouri M, Slassi I, et al. Duchenne and Becker muscular dystrophy: Contribution of a molecular and immunohistochemical analysis in diagnosis in Morocco. J Biomed Biotechnol. 2009;2009:325210. https://doi.org/10.1155/2009/325210 PMid:19461958 DOI: https://doi.org/10.1155/2009/325210
Janssen B, Hartmann C, Scholz V, Jauch A, Zschocke J. MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: Potential and pitfalls. Neurogenetics. 2005;6(1):29-35. https://doi.org/10.1007/s10048-004-0204-1 PMid:15655674 DOI: https://doi.org/10.1007/s10048-004-0204-1
Manjunath M, Kiran P, Preethish-Kumar V, Nalini A, Singh R, Gayathri N. A comparative study of mPCR, MLPA, and muscle biopsy results in a cohort of children with Duchenne muscular dystrophy: A first study. Neurol India. 2015;63(1):58-62. https://doi.org/10.4103/0028-3886.152635 PMid:25751470 DOI: https://doi.org/10.4103/0028-3886.152635
Suriyonplengsaeng C, Dejthevaporn C, Khongkhatithum C, Sanpapant S, Tubthong N, Pinpradap K, et al. Immunohistochemistry of sarcolemmal membrane-associated proteins in formalin-fixed and paraffin-embedded skeletal muscle tissue: A promising tool for the diagnostic evaluation of common muscular dystrophies. Diagn Pathol. 2017;12(1):1-10. https://doi.org/10.1186/s13000-017-0610-y PMid:28219397 DOI: https://doi.org/10.1186/s13000-017-0610-y
Bushby K, Finkel R, Birnkrant DJ, Case LE, Clemens PR, Cripe L, et al. Diagnosis and management of Duchenne muscular dystrophy, Part 1: Diagnosis, and pharmacological and psychosocial management. Lancet Neurol. 2010;9(1):77-93. https://doi.org/10.1016/S1474-4422(09)70271-6 PMid:19945913 DOI: https://doi.org/10.1016/S1474-4422(09)70271-6
Vogel H, Zamecnik J. Diagnostic immunohistology of muscle diseases. J Neuropathol Exp Neurol. 2005;64(3):181-93. https://doi.org/10.1093/jnen/64.3.181 PMid:15804049 DOI: https://doi.org/10.1093/jnen/64.3.181
Hoshino S, Ohkoshi N, Watanabe M, Shoji S. Immunohistochemical staining of dystrophin on formalin-fixed paraffin-embedded sections in Duchenne/Becker muscular dystrophy and manifesting carriers of Duchenne muscular dystrophy. Neuromuscul Disord. 2000;10(6):425-9. https://doi.org/10.1016/S0960-8966(99)00116-9 PMid:10899449 DOI: https://doi.org/10.1016/S0960-8966(99)00116-9
Sheriffs IN, Rampling D, Smith VV. Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy. J Clin Pathol. 2001;54(7):517-20. http://doi.org/10.1136/jcp.54.7.517 PMid:11429422 DOI: https://doi.org/10.1136/jcp.54.7.517
Nishida A, Kataoka N, Takeshima Y, Yagi M, Awano H, Ota M, et al. Chemical treatment enhances skipping of a mutated exon in the dystrophin gene. Nat Commun. 2011;2(1):308. https://doi.org/10.1038/ncomms1306 DOI: https://doi.org/10.1038/ncomms1306
Gibbs EM, Barthélémy F, Douine ED, Hardiman N, Shieh PB, Khanlou N, et al. Large in-frame 5’ deletions in DMD associated with mild Duchenne muscular dystrophy: Two case reports and a review of the literature. Neuromuscul Disord. 2019;29(11):863-73. https://doi.org/10.1016/j.nmd.2019.09.009 PMid:31672265 DOI: https://doi.org/10.1016/j.nmd.2019.09.009
Nadkarni J, Dastur R, Gaitonde P, Khadilkar S. Becker muscular dystrophy in Indian patients: Analysis of dystrophin gene deletion patterns. Neurol India. 2008;56(3):374. https://doi.org/10.4103/0028-3886.40961 PMid:18974567 DOI: https://doi.org/10.4103/0028-3886.40961
Nowak KJ, Davies KE. Duchenne muscular dystrophy and dystrophin: Pathogenesis and opportunities for treatment. EMBO Rep. 2004;5(9):872-6. https://doi.org/10.1038/sj.embor.7400221 PMid:15470384 DOI: https://doi.org/10.1038/sj.embor.7400221
Echigoya Y, Lee J, Rodrigues M, Nagata T, Tanihata J, Nozohourmehrabad A, et al. Mutation types and aging differently affect revertant fiber expansion in dystrophic Mdx and Mdx52 mice. PLoS One. 2013;8(7):e69194. https://doi.org/10.1371/journal.pone.0069194 PMid:23894429 DOI: https://doi.org/10.1371/journal.pone.0069194
Schwartz M, Dunø M. Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent probe amplification method.Genetic Testing. 2004;8(4):361-7. https://doi.org/10.1089/gte.2004.8.361 PMid:15684864 DOI: https://doi.org/10.1089/gte.2004.8.361
Todorova A, Todorov T, Georgieva B, Lukova M, Guergueltcheva V, Kremensky I, et al. MLPA analysis/ complete sequencing of the DMD gene in a group of Bulgarian Duchenne/Becker muscular dystrophy patients. Neuromuscul Disord. 2008;18(8):667-70. https://doi.org/10.1016/j.nmd.2008.06.369 DOI: https://doi.org/10.1016/j.nmd.2008.06.369
Grunau G, Linn S. Commentary: Sensitivity, specificity, and predictive values: Foundations, pliabilities, and pitfalls in research and practice. Front Public Health. 2018;6:1-4. https://doi.org/10.3389/fpubh.2018.00256 DOI: https://doi.org/10.3389/fpubh.2018.00256
Wong HB, Lim GH. Measures of diagnostic accuracy: Sensitivity, specificity, PPV and NPV. Proc Singapore Healthc. 2011;20(4):316-8. DOI: https://doi.org/10.1177/201010581102000411
Werneck LC, Scola RH, Henrique G, Maegawa B. Comparative analysis of PCR-deletion detection and immunohistochemistry in Brazilian Duchenne and Becker muscular dystrophy patients. Am J Med Genet. 2001;103(2):115-20. https://doi.org/10.1002/ajmg.1508 PMid:11568916 DOI: https://doi.org/10.1002/ajmg.1508
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