Exon-skipped dystrophins for treatment of Duchenne muscular dystrophy: Mass spectrometry mapping of most exons and cooperative domain designs based on single molecule mechanics

Abstract Background Relative haplotype dosage (RHDO) approach has been applied in noninvasive prenatal diagnosis (NIPD) of Duchenne muscular dystrophy (DMD). However, the RHDO procedure is relatively complicated and the parental haplotypes need to be constructed. Furthermore, it is not suitable for the diagnosis of de novo mutations or mosaicism in germ cells. Here, we investigated NIPD of DMD using a relative mutation dosage (RMD)-based approach—cell-free DNA Barcode-Enabled Single-Molecule Test (cfBEST), which has not previously been applied in the diagnosis of exon deletion. Methods Five DMD families caused by DMD gene point mutations or exon deletion were recruited for this study. After the breakpoints of exon deletion were precisely mapped with multiple PCR, the genotypes of the fetuses from the five DMD families were inferred using cfBEST, and were further validated by invasive prenatal diagnosis. Results The cfBEST results of the five families indicated that one fetus was female and did not carry the familial molecular alteration, three fetuses were carriers and one was male without the familial mutation. The invasive prenatal diagnosis results were consistent with those of the cfBEST procedure. Conclusion This is the first report of NIPD of DMD using the RMD-based approach. We extended the application of cfBEST from point mutation to exon deletion mutation. The results showed that cfBEST would be suitable for NIPD of DMD caused by different kinds of mutation types.

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Muscle metabolic remodelling patterns in Duchenne muscular dystrophy revealed by ultra-high-resolution mass spectrometry imaging

Scientific Reports ◽

10.1038/s41598-021-81090-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ivana Dabaj ◽

Justine Ferey ◽

Florent Marguet ◽

Vianney Gilard ◽

Carole Basset ◽

...

Keyword(s):

Mass Spectrometry ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Molecular Mechanisms ◽

High Resolution Mass Spectrometry ◽

Phospholipid Metabolism ◽

Ion Cyclotron Resonance ◽

Unbiased Manner ◽

Phosphatidic Acids ◽

Muscle Biopsies

AbstractDuchenne muscular dystrophy (DMD) is a common and severe X-linked myopathy, characterized by muscle degeneration due to altered or absent dystrophin. DMD has no effective cure, and the underlying molecular mechanisms remain incompletely understood. The aim of this study is to investigate the metabolic changes in DMD using mass spectrometry-based imaging. Nine human muscle biopsies from DMD patients and nine muscle biopsies from control individuals were subjected to untargeted MSI using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry. Both univariate and pattern recognition techniques have been used for data analysis. This study revealed significant changes in 34 keys metabolites. Seven metabolites were decreased in the Duchenne biopsies compared to control biopsies including adenosine triphosphate, and glycerophosphocholine. The other 27 metabolites were increased in the Duchenne biopsies, including sphingomyelin, phosphatidylcholines, phosphatidic acids and phosphatidylserines. Most of these dysregulated metabolites are tightly related to energy and phospholipid metabolism. This study revealed a deep metabolic remodelling in phospholipids and energy metabolism in DMD. This systems-based approach enabled exploring the metabolism in DMD in an unprecedented holistic and unbiased manner with hypothesis-free strategies.

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Lipid imaging by gold cluster time-of-flight secondary ion mass spectrometry: application to Duchenne muscular dystrophy

The Journal of Lipid Research ◽

10.1194/jlr.m500058-jlr200 ◽

2005 ◽

Vol 46 (7) ◽

pp. 1388-1395 ◽

Cited By ~ 102

Author(s):

David Touboul ◽

Alain Brunelle ◽

Frédéric Halgand ◽

Sabine De La Porte ◽

Olivier Laprévote

Keyword(s):

Mass Spectrometry ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Secondary Ion Mass Spectrometry ◽

Gold Cluster ◽

Time Of Flight ◽

Ion Mass Spectrometry ◽

Secondary Ion ◽

Mass Spectrometry Application

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Dystrophin and mini-dystrophin quantification by mass spectrometry in skeletal muscle for gene therapy development in Duchenne muscular dystrophy

Gene Therapy ◽

10.1038/s41434-021-00300-7 ◽

2021 ◽

Author(s):

Vahid Farrokhi ◽

Jason Walsh ◽

Joe Palandra ◽

Joanne Brodfuehrer ◽

Teresa Caiazzo ◽

...

Keyword(s):

Mass Spectrometry ◽

Gene Therapy ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Biceps Femoris ◽

Dystrophin Gene ◽

Limit Of Quantification ◽

Post Dose ◽

Validation Data ◽

Dystrophin Expression

AbstractDuchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disorder caused by mutations in the DMD gene, leading to severe reduction or absence of the protein dystrophin. Gene therapy strategies that aim to increase expression of a functional dystrophin protein (mini-dystrophin) are under investigation. The ability to accurately quantify dystrophin/mini-dystrophin is essential in assessing the level of gene transduction. We demonstrated the validation and application of a novel peptide immunoaffinity liquid chromatography–tandem mass spectrometry (IA-LC-MS/MS) assay. Data showed that dystrophin expression in Becker muscular dystrophy and DMD tissues, normalized against the mean of non-dystrophic control tissues (n = 20), was 4–84.5% (mean 32%, n = 20) and 0.4–24.1% (mean 5%, n = 20), respectively. In a DMD rat model, biceps femoris tissue from dystrophin-deficient rats treated with AAV9.hCK.Hopti-Dys3978.spA, an adeno-associated virus vector containing a mini-dystrophin transgene, showed a dose-dependent increase in mini-dystrophin expression at 6 months post-dose, exceeding wildtype dystrophin levels at high doses. Validation data showed that inter- and intra-assay precision were ≤20% (≤25% at the lower limit of quantification [LLOQ]) and inter- and intra-run relative error was within ±20% (±25% at LLOQ). IA-LC-MS/MS accurately quantifies dystrophin/mini-dystrophin in human and preclinical species with sufficient sensitivity for immediate application in preclinical/clinical trials.

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