An article describing a new protocol for the molecular diagnosis of Duchenne muscular dystrophy (DMD) was published in the journal Current Protocols. The detailed protocol includes a sequencing assay based on next-generation sequencing, which considers sequence and copy number variants in the DMD gene.

Unlike the traditional strategy for DMD molecular diagnosis, which involves an initial screening for deletions or duplications then a full-sequence analysis of the DMD gene, the new protocol comprises a single assay, which promises to be quicker and less expensive.

The new assay involves 15 steps that each include multiple steps, resulting in 105 total steps that can be completed within 4 days.

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These 15 steps are: shearing the DNA, assessing the quality of sheared DNA, preparing a library, amplifying the library, purifying the amplified library sample, assessing the quality of the amplified library sample, performing the hybridization, capturing and washing the hybridized samples, purifying the captured sample, amplifying the purified captured samples, purifying the amplified captured samples, assessing the quality of the amplified captured sample, sequencing the prepared samples, analyzing the sequence variant data, and analyzing the copy number variant data.

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According to the authors of the study, the new assay could “establish a molecular diagnosis in around 86% of DMD cases.” The remaining 13% of cases may include deep intronic variants, silent variants, translocations or other structural rearrangements, and errors in the regulatory regions of the gene.

DMD is caused by a mutation in the DMD gene, which is the largest known human gene. The vast spectrum of disease-causing mutations in the gene, including intragenic deletions and duplications, point mutations, and other sequence variants, make molecular testing of DMD particularly challenging.


Nallamilli BRR, Guruju N, Jump V, Liu R, Hegde M. Molecular diagnosis of Duchenne muscular dystrophy using single NGS-based assay. Curr Protoc. 2023;3(2):e669. doi:10.1002/cpz1.669