Dystrophin as a Diagnostic Marker in Duchenne and Becker Muscular Dystrophy. Correlation of Immunofluorescence and Western Blot

Abstract

Dystrophin is the gene product of the Duchenne (DMD) and Becker (BMD) muscular dystrophy gene locus on the short arm of the X chromosome. Complete lack of dystrophin is pathognomonic for DMD and variable changes of the molecule may be observed in the milder allelic form of BMD. In the present study the two methods available for dystrophin assessment, immunofluorescence detections on cryosections (IF) and Western blotting (WB) were systematically compared using polyclonal and monoclonal antibodies to various regions along the dystrophin molecule. A total of 95 patients with DMD or BMD were investigated including two female patients. Dystrophin assessment revealed abnormal abundance and/or distribution in all 95 patients with DMD or BMD. Only trace amounts of dystrophin were detected in 29% of the DMD patients and complete lack of dystrophin was found in 71%. In two females with DMD but with normal karyotype single dystrophin-positive fibres were found among more than 90% negative fibres. Out of 26 patients with BMD 19 (73%) had a dystrophin molecule of abnormal molecular weight. The results of IF were largely compatible with those from WB but differences were also observed, e.g. one barely symptomatic BMD patient with dystrophin of increased molecular weight showed normal IF. Out of four carriers of BMD three showed evidence of reduced dystrophin immunostaining in some muscle fibres. In 20 other patients limb girdle muscualar dystrophy with "Duchenne-like" or "Becker-like" phenotype was suspected because dystrophin showed normal abundance and distribution. Focal discontinuity of muscle cell-surface dystrophin staining was observed in one patient with a congenital, autosomal recessive muscular dystrophy and in one out of five patients with polymyositis/dermatomyositis. The study emphasizes the need for, and value of, dystrophin assessment in every case of suspected BMD or DMD.

Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins

“…In a study performed by Voit et al, 40 up to 30% of DMD patients had some dystrophin expression as detected by Western blots and immunohistochemistry. Other groups have also reported the incidence of revertant fibres in 35-70% of DMD patients.…”

Duchenne muscular dystrophy

“…Immunoreactivity to carboxy-terminal antibodies however is absent in DMD and is therefore useful in differentiating DMD from BMD. [77][78][79] Western blot analysis allows quantification of the amount of dystrophin protein as well assessment of size of the protein present. In DMD less than 5% of the normal quantity of dystrophin is present when carboxy-terminal antibodies are used, whilst up to 25% of normal dystrophin levels is seen with the use of rod domain antibodies.…”

Functional significance of dystrophin positive fibres in Duchenne muscular dystrophy.

“…"'7 Labelling may occur even with carboxy terminal antibodies,'4 16 and serial sections labelled with antibodies that recognise epitopes before and after frame shifting deletions may show immunoreactivity with the same few fibres or groups of fibres. '8 '9 This indicates that such muscle from patients with DMD contains very low concentrations of dystrophin that must have been synthesised from mRNA in which the reading frame had been restored.8 20 As the presence of in-frame dystrophin molecules is associated with milder clinical phenotypes, the aim ofthis report is to answer the question: 'is the level ofdystrophin synthesis in DMD patients too low to have any functional significance?'…”