Diagnostic value of dystrophin immunostaining in the diagnosis of duchenne and becker muscular dystrophy patients

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. AIM: The objectives of the study were 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 2 × 2 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. © 2021 Shinta Andi Sarasati, Kristy Iskandar, Maria Alethea Septinastiti, Rusdy Ghazali Malueka, Ery Kus Dwianingsih.