Figure 1.
Characterization of the double band corresponding to dysferlin observed in CD14+ PBM by WB.
A) Dysferlin siRNA efficiently reduced both dysferlin bands in PBM. Representative WB of monocytes from a healthy donor treated with siRNA showed that siDYSF efficiently reduced the two bands corresponding to dysferlin. On the right, quantification of WB from silenced monocyte samples, corresponding to four independent experiments, showed that when dysferlin was knocked down, levels diminished from 100% in Control (C) and 104.2±11.3% in siMYOF to 58.4±14.8% with siDYSF1 (p<0.05) and 72.2±9.8% with siDYSF2 (p<0.05), error bars indicate standard desviation. B) Schematic overview of the dysferlin protein and the epitopes recognized by F4, H7 and Hamlet antibodies. The three affinity binders together cover the complete dysferlin protein sequence. C) Both F4 and H7 antibodies can immunoprecipitate the dysferlin doublet from PBM. Bound (B) and non-bound (NB) fractions were analyzed by WB for dysferlin. The arrow denotes the dysferlin doublet observed in the bound fraction using F4 and H7 and in the non-bound fraction using an antibody against β-amyloid.
Table 1.
Summary of dysferlin myopathy patients: Correlation with protein expression in PBM, skeletal muscle and mutations.
Figure 2.
Correlation between dysferlin expression in skeletal muscle, detected by IH and WB, and WB of PBM.
A) Healthy control with normal expression of dysferlin in PBM: Dysferlin is expressed in the sarcolemma of muscle fibres. WB of skeletal muscle and PBM showed normal levels of dysferlin. B) Dysferlinopathy patient (P_3): Dysferlin is absent in sarcolemma by IH, WB and in PBM. C) Patient with LGMD2A (C_1) showing abnormal expression of dysferlin in skeletal muscle by IH but normal levels in skeletal muscle and PBM WB. D) Symptomatic carrier of one mutation in DYSF: muscle IH and WB show reduced expression that was confirmed in PBM. HMZ: homozygous.
Table 2.
Summary of patients with normal expression of dysferlin included in this study.
Figure 3.
Parallelism between dysferlin expression in PBM and in skeletal muscle.
Linear association between dysferlin expression in skeletal muscle (X axis) and in PBM (Y axis) were observed (R2 = 0.9766, p<0.001). Black diamonds show dysferlin expression in pathological controls and white diamonds show dysferlin expression in dysferlinopathy patients.
Figure 4.
Dysferlin staining patterns in muscle biopsy from dysferlinopathy patients and from other muscle diseases.
Muscle biopsies from dysferlinopathy patients show three staining patterns: A) Normal dysferlin staining in a pathological control muscle (C_20 with calf atrophy in Table 2). B) Total absence of dysferlin expression at sarcolemma and sarcoplasm (P_2 with Miyoshi myopathy in Table 1). C) reduced sarcolemmal staining in all muscles (C_8 with Nonaka myopathy in Table 2). D) A sarcoplasm granular pattern in scattered fibers was observed in some dysferlinopathy patients (P_7 with LGMD2B) in Table 1) E) Increased cytoplasmic staining of dysferlin in all muscle fibres (C_16 with LGMD in Table 2). F) A patchy staining of dysferlin was observed in the sarcolemma in some muscle fibres (P_15 with Miyoshi myopathy in Table 1). G) Some muscle biopsies had increased dysferlin staining in the cytoplasm of some fibers (P_13 with PM in Table 2).