Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens

Taylor, Laura E.; Kaminoh, Yuuki; Rodesch, Christopher K.; Flanigan, Kevin M.

doi:10.1111/j.1365-2990.2012.01250.x

Cited by 54 publications

(82 citation statements)

References 13 publications

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“…All image parameters including pinhole size, detector gain, amplifier offset, amplifier gain, and laser intensity were first set for normal control tissue, and the same setting used for all samples imaged on a given day. 10 Both control and patient sample images were obtained at the same day after the staining. For each sample, 5 nonoverlapping sections were acquired.…”

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Reduced neurofilament expression in cutaneous nerve fibers of patients with CMT2E

et al. 2015

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Objective: To investigate the effects of NEFL Glu396Lys mutation on the expression and assembly of neurofilaments (NFs) in cutaneous nerve fibers of patients with Charcot-Marie-Tooth disease type 2E (CMT2E).Methods: A large family with CMT2E underwent clinical, electrophysiologic, and skin biopsy studies. Biopsies were processed by indirect immunofluorescence (IF), electron microscopy (EM), and Western blot analysis. Results:The clinical features demonstrated intrafamilial phenotypic variability, and the electrophysiologic findings revealed nerve conductions that were either slow or in the intermediate range. All patients had reduced or absent compound muscular action potential amplitudes. Skin biopsies showed axons labeled with the axonal markers protein gene product 9.5 and a-tubulin, but not with NFs. The results of Western blot analysis were consistent with those of IF, showing reduced or absent NFs and normal expression of a-tubulin. EM revealed clusters of regenerated fibers, in absence of myelin sheath abnormalities. Both IF and EM failed to show NF aggregates in dermal axons. The morphometric analysis showed a smaller axonal caliber in patients than in controls. The study of the nodal/paranodal architecture demonstrated that sodium channels and Caspr were correctly localized in patients with CMT2E.Conclusions: Decrease in NF abundance may be a pathologic marker of CMT2E. The lack of NF aggregates, consistent with prior studies, suggests that they occur proximally leading to subsequent alterations in the axonal cytoskeleton. The small axonal caliber, along with the normal molecular architecture of nodes and paranodes, explain the reduced velocities detected in patients with CMT2E. Our results also demonstrate that skin biopsy can provide evidence of pathologic and pathogenic abnormalities in patients with CMT2E. Charcot-Marie-Tooth disease (CMT) is a group of peripheral neuropathies associated with mutations in more than 80 distinct genes. CMT is divided into different forms based on the pattern of inheritance and neurophysiology. Electrophysiologic studies allow for classification of CMT into demyelinating (CMT1) and axonal (CMT2) forms.1 The classification of CMT has been further divided into subtypes, identified by letters, as defined by the mutated gene.2 CMT2E refers to autosomal dominant CMT2 caused by mutations in the neurofilament light chain (NEFL) gene. Some patients with NEFL mutations have slow conduction velocities, in the "demyelinating" range, and are characterized as having CMT1F. Patients with CMT2E and CMT1F are clinically heterogeneous and the age at onset ranges from infancy, typically with severe impairment, to adulthood with milder impairment. Usually those with slowed conductions present early either in infancy with delayed motor milestones or in late childhood/early adolescence.

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Reduced neurofilament expression in cutaneous nerve fibers of patients with CMT2E

et al. 2015

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“…With the advent of dystrophin restoring strategies and the need for meticulous evaluation of therapies, improvements in dystrophin detection and quantification have become an imperative, and two groups have published detailed methods for the unbiased quantification of dystrophin immunofluorescent expression [20,21]. Detailed dystrophin quantification using one of these [21] along with clinical correlations in BMD patients clearly indicate that internally deleted dystrophin isoforms have the capacity to confer marked clinical benefits to individuals with DMD [22].…”

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confidence: 99%

“…The sensitivity of current immunofluorescent quantification methods [20,21] should allow reproducible fold-change quantification at even low levels of baseline expression, but whether dystrophin expression is measured by western blotting or by immunohistochemistry techniques, it is logical that fold changes and not fractional increases in dystrophin production above baseline will be needed to ameliorate disease progression in DMD. The minimally sufficient fold change necessary for functional improvement is unlikely to be universally definable; it may differ for different therapies, as it may be dependent upon the induction of the specific dystrophin isoform resulting from therapy, the duration of treatment, and other factors.…”

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Dystrophin as a therapeutic biomarker: Are we ignoring data from the past?

Wilton¹,

Fletcher²,

Flanigan³

2014

Neuromuscular Disorders

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“…For averages or multiple measurements in sections from a manifesting carrier, a clear segregation of measurements is immediately evident using the original method 10 , which is lost with the new method (Figure 1a and b). 2 Similarly, two patients from a recent clinical trial have almost identical levels of dystrophin when assessed using the new Author's pre-print version. Original version can be found at http://goo.gl/husrE.…”

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confidence: 99%

“…findings on a new method for rapid dystrophin quantification in Duchenne muscular dystrophy (DMD) 2 . Although of interest, we believe that caution is required in the interpretation of dystrophin measurements obtained using this new technique.…”

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Measuring dystrophin—faster is not necessarily better

et al. 2012

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A Research Highlight in the February issue (A novel imaging method to quantify low levels ) 1 presented of dystrophin in Duchenne muscular dystrophy. Nat. Rev. Neurol. 8, 120;2012 findings on a new method for rapid dystrophin quantification in Duchenne muscular dystrophy (DMD) 2 . Although of interest, we believe that caution is required in the interpretation of dystrophin measurements obtained using this new technique.DMD is caused by the deficit of dystrophin protein at sarcolemma of muscle fibres 3 . Quantification of dystrophin on muscle biopsies is the main diagnostic test for DMD when genetic testing is unavailable. Several therapeutic approaches in DMD aim to restore dystrophin expression: redirection of splicing with antisense oligonucleotides; 4, 5 gene therapy 6 ; stem cell therapy; 7 and nonsense mutation read-through. 8,9 Precise quantification of dystrophin protein in muscle biopsies taken before and after treatment is crucial to evaluate the biochemical success of the therapeutic intervention.Until recently, counting dystrophin positive fibres or western blotting were the only quantitative methods available, but researchers preparing for the first trials developed a method to sensitively quantify dystrophin and other associated proteins in the muscle fibre sarcolemma using only two muscle sections per antibody. 10 The method uses intensity measurements from fluorescently labelled dystrophin antibodies and spectrin labelling as a normalising factor. The technique greatly advanced dystrophin quantification owing to its sensitivity, requirement for very little sample, capacity to confirm the correct localization of the protein at sarcolemma, and accessibility to most pathology laboratories. Despite being labour intensive, this method has been used in the analysis of several clinical trials, 4, 5 and in human 11-13 and mouse 14, 15 studies.Aided by a new spectrin antibody that enabled immunostaining for dystrophin and spectrin on the same section, researchers have been able to automate this technique 2 , which should accelerate the analysis of muscle biopsies in ongoing clinical trials. Despite the unequivocal advance that this method entails, one should note the potential drawbacks. One important aspect is that the original method involves collection of up to 40 data points per section, each corresponding to a muscle fibre, whereas the recent modification involves collecting an average dystrophin intensity of the whole image. For averages or multiple measurements in sections from a manifesting carrier, a clear segregation of measurements is immediately evident using the original method 10 , which is lost with the new method (Figure 1a and b). 2 Similarly, two patients from a recent clinical trial have almost identical levels of dystrophin when assessed using the new

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Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens

Abstract: This method offers a robust and reliable method of biomarker detection for trials of DMD therapies.

Cited by 54 publications

References 13 publications

Reduced neurofilament expression in cutaneous nerve fibers of patients with CMT2E

Reduced neurofilament expression in cutaneous nerve fibers of patients with CMT2E

Dystrophin as a therapeutic biomarker: Are we ignoring data from the past?

Measuring dystrophin—faster is not necessarily better

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