Summary of α-DG glycosylation as assessed by flow cytometry in 21 patient fibroblasts, three healthy controls, and seven pathological controls.

<p>The specific gene, mutation, phenotype, MFI of the IIH6 positive cells ± SEM, percentage of cells positive for the IIH6 epitope ± SEM, iMFI value ± SEM, N value, P value, and muscle α-DG IIH6 description is listed for each fibroblast cell line analysed. P values are the result of an unpaired t-test comparing the MFI, percentage of cells positive for the IIH6 epitope, or iMFI values of each fibroblast cell line to the respective values of the three healthy controls (C1, C2, C3). Muscle α-DG IIH6 description is the summary from the patient report of how the skeletal muscle α-DG glycosylation appeared by immunohistochemistry. iMFI is defined as iMFI = (MFI)(<i>P</i>), where P is the percentage of cells positive for the IIH6 epitope <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068958#pone.0068958-Darrah1" target="_blank">[43]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068958#pone.0068958-Shooshtari1" target="_blank">[44]</a>. Abbreviations are as follows: DMD, Duchenne muscular dystrophy; BMD, Becker muscular dystrophy; LGMD, Limb-girdle muscular dystrophy; MEB, Muscle-eye-brain disease; MDC1C, Muscular dystrophy type 1 C; CMD, Congenital muscular dystrophy; FCMD, Fukuyama congenital muscular dystrophy; LGMD-CRB, limb girdle muscular dystrophy with cerebellar involvement <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068958#pone.0068958-Cirak1" target="_blank">[11]</a>; MR, mental retardation; NS, non-significant. * = Sections evaluated with the VIA4-1 antibody.</p

Comparison of the mean fluorescence intensity (MFI) of α-DG glycosylation in patient fibroblasts to respective patient phenotypic severity and skeletal muscle α-DG IIH6 immunolabelling.

<p>(A) Fibroblasts from patients with relatively mild phenotypes (LGMD, LGMD-like without brain involvement, includes P1, P5, P6, P12, P13, and P16 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068958#pone-0068958-t001" target="_blank">table 1</a>) have a significantly (p = 0.008) higher average MFI value (61.5±12.1) than fibroblasts from patients with more severe dystroglycanopathy phenotypes (MEB, FCMD, MEB/FCMD-like, includes P2, P7, P8, P9, P10, P14, P15, P18, P19, P20 with an MFI average of 31.0±2.9). (B) In patient fibroblasts, a decrease in the MFI of IIH6 obtained by flow cytometry concomitant with a decrease in the intensity of immunolabelling in skeletal muscle sections was observed. Skeletal muscle sections which were described as ‘absent’ or ‘markedly reduced’ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068958#pone-0068958-t001" target="_blank">table 1</a>) for IIH6 yielded an average MFI from the respective fibroblasts of 30.7±3.1 (n = 9). Sections described as ‘reduced’ for IIH6 yielded an average MFI value of 38.3±4.0 (n = 7) from the respective fibroblasts. Finally, sections which were described as ‘normal’ (or almost normal) yielded MFI values of 76.7±7.7 (n = 5) from the respective fibroblasts. There is a significant difference in the MFI between both groups reduced for skeletal muscle α-DG IIH6 immunolabelling and the group described as normal or almost normal (p<0.0001 for absent/markedly reduced, p = 0.0007 for reduced). *** = p<0.001, ** = p<0.01 (unpaired t-test). In all cases values are described as mean fluorescence intensity ± standard error of the mean (SEM).</p

Dystrophin quantification in a population of myofibres identified in entire muscle sections performing the double labelling anti-dystrophin ab15277 and anti-spectrin.

<p>Representative images of entire muscle sections stained and acquired by the Axio Scan slide scanner and processed with Definens algorithm derived from a control (a) and from a DMD patient (b). Graph of representative dystrophin quantification in the fibre population of a control, a DMD and a BMD muscle sample (c). Dystrophin quantification was plotted as cumulative fibre count (%) on the primary y axis. The blue dashed line represents the dystrophin intensity distribution of a representative control sample (control 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194540#pone.0194540.t001" target="_blank">Table 1</a>), the green dashed line represents the BMD sample and the DMD sample is represented by the red dashed line. The distribution curve of the absolute dystrophin fibre count for the control sample is represented by the blue peak, for the BMD is expressed by the green peak and the DMD fibre population curve is represented by the red peak. Fibres analysed in this representative muscle sections were: ~ 14500 for the control, ~ 10000 for the BMD and ~ 2700 for the DMD muscle section.</p> <p>DMD: Duchenne Muscular Dystrophy; BMD: Becker Muscular Dystrophy; i.v: intensity values.</p

Algorithm development for identifying myofibres.

<p>Spectrin or laminin allow fibre identification within the muscle section. The algorithm was instructed to recognize the tissue within the image (a) and to subsequently recognize the myofibres within the section (b). The algorithm then generated a mask considering only structures presenting fibre characteristics (i.e. excluding nerves, spindles, blood cells, folded muscle tissue and connective areas (c). The mask generated by the algorithm included only the sarcolemma compartment of myofibres and clearly defined fibre rims (d).</p

Spectrin and laminin mean intensity in muscle sections of DMD, BMD and control patients.

<p>Muscle sections were cut from two paediatric controls, two DMD and two BMD patients. Two biological replicate experiments were performed and for each experiment two sections per sample were stained with anti-spectrin or anti-laminin and acquired by the Axio Scan slide scanner. The Definens script extrapolated spectrin or laminin intensities for each individual myofibre identified within the section. Protein intensities data for spectrin (a) or for laminin (b) were grouped per category (CTR, DMD and BMD) as mean±SEM. The Mann Whitney test revealed significant differences between CTR, DMD and BMD for both spectrin (a) and laminin (b) fluorescent intensities (***, p<0.001). (CTR: control; DMD: Duchenne muscular dystrophy; BMD: Becker muscular dystrophy; SEM: standard error of the mean).</p

Antibodies used on muscle sections.

<p>Antibodies used on muscle sections.</p

Dystrophin expression in different muscle types obtained from paediatric controls.

<p>Ten muscle blocks derived from different muscle groups obtained from seven controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194540#pone.0194540.t001" target="_blank">Table 1</a>) were analysed performing anti-dystrophin ab15277/anti-spectrin and anti-dystrophin MANDYS106/anti-laminin stainings. Entire muscle sections were acquired by the Axio Scan slide scanner and processed by the Definiens algorithm, exploiting spectrin or laminin staining for fibre identification. Dystrophin was quantified in each individual fibre using either anti-dystrophin ab15277 (Fig 4a) or MANDYS106 (Fig 4b) intensities plotted as arbitrary units. We acquired a different number of myofibres (22,000 fibres for control 3 to 5,000 fibres for control 5) per section depending on the cross sectional area.</p

Intensity dystrophin range in muscle sections of DMD/BMD patients and controls.

<p>Intensity dystrophin range in muscle sections of DMD/BMD patients and controls.</p

Labelling variability in different biological replicates of controls.

<p>Labelling variability in different biological replicates of controls.</p

Variability in dystrophin quantification in independent immunostaining and dystrophin signal detection stability across the time.

<p>Double staining with different antibody combinations were performed (anti-dystrophin ab15277/anti spectrin (a) and anti-dystrophin MANDYS106/anti-laminin (b)). Sections were cut and stained immediately (A). Muscle blocks were kept at -80°C for one month and then two section sets were cut and again stained immediately. One set of these stained sections were acquired immediately (B) whereas the other stained section set was kept at 4°C for three months and then acquired (C) in order to evaluate the fluorescent signal stability of dystrophin staining after slides long time storage. Dystrophin intensities were quantified per each individual fibre and were plotted as scatter plots (mean±SEM) in arbitrary units (***p< 0.001, Mann Whitney test). Dystrophin intensity dynamic range and number of fibres acquired per experiment are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194540#pone.0194540.t003" target="_blank">Table 3</a>.</p