The clinical characteristics of Duchenne muscular dystrophy (DMD) are well described in young male patients. Physicians that are able to identify these clinical characteristics early can order additional diagnostic tests to validate their suspicions. A management plan can then be immediately created.

Read more about DMD etiology

Researchers from China described the case of a young patient with DMD who had long-read whole-genome sequencing performed to diagnose the precise molecular cause of his disease. This case study was published in the Annals of Clinical and Translational Neurology and is the subject of our article today. 

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The Case Study

The patient in this case study is a 9-year-old boy who presented with a classical DMD phenotype; he was unable to walk unaided and had obvious tendon contractures and a waddling gait. He also demonstrated toe walking. 

When the patient was 6.5 years of age, he presented to the Peking University First Hospital in Beijing with a history of delayed motor milestones and proximal muscle weakness since he was 2 years old. Physical examinations corroborated his complaints: he had a positive Gowers’ sign, limb-girdle muscle weakness, mild bilateral tendon contractures, and calf hypertrophy. 

When he was seen again at 9.5 years of age, serum creatinine kinase levels were highly elevated, with a range of 6510 to 11,896 IU/L (normal range: 25–170 IU/L). Muscle magnetic resonance imaging (MRI) demonstrated the trefoil with single fruit sign at the proximal thigh level, which is highly suggestive of dystrophinopathies. In view of his DMD phenotype, routine DNA testing was carried out, but no casual variants were identified. 

A muscle biopsy was subsequently performed, revealing a dystrophin pattern and an absence of dystrophin expression; this sufficed as a molecular diagnosis of DMD. To proceed further in their investigations, researchers conducted a dystrophin messenger RNA (mRNA) analysis to look for possible aberrant transcripts. They found that the second to 15th complementary DNA (cDNA) fragments were absent, suggesting possible skipping of exons 8 to 51. This was theorized to have caused a frameshift and premature termination codon that occurred 33 codons downstream of exon 52, which would be consistent with the absence of dystrophin expression caused by nonsense-mediated decay. 

Read more about DMD prognosis

Encouraged by these findings, researchers proceeded to perform short-read sequencing of the entire DMD gene to look for variants that could have caused exon skipping. The researchers did not identify any causative intronic variants, so they proceeded to perform long-read sequencing of the DMD gene. A possible inversion involving exons 8 to 51 was indicated, with 10 reads with an average length of 1938 bp. However, researchers could only validate the 3′ breakpoint region according to the information gathered from the 10 reads. The low depth and coverage of the 5′ breakpoint region meant that it was simply not informative enough for further Sanger validation, which researchers theorized could be due to a large deletion event around the 5′ breakpoint region. 

However, they did not give up. “For further precise identification and validation of the possible inversion, we performed whole-genome long-read sequencing using the Nanopore PromethION (Oxford Nanopore, Oxford, UK) sequencer,” the authors of the study stated. They then found 13 reads with an average length of 62,738 bp, indicating not only the possible inversion around the 5′ breakpoint region, but also a deletion event. With further aid from Sanger sequencing, they were finally able to confirm the exact sequence of the 5′ breakpoint region and concluded that it was indeed a deletion-insertion event.

Intronic Variants in DMD 

“The DMD gene, spanning over 2.5 Mb, is the largest gene described in the human genome, more than 99% of which is the intronic region,” the authors of the study wrote. The structural complexity of DMD complicates efforts to identify the full spectrum of DMD variants. Therefore, there must be flexibility in our genetic approaches when routine testing proves inadequate.

Researchers in this case study performed a dystrophin mRNA analysis due to previous reports demonstrating that deep intronic variants could be identified through this methodology. In the case of this patient, a dystrophin mRNA analysis showed an informative aberrant splicing event. Subsequent DMD gene sequencing demonstrated a possible large-scale inversion in DMD. Researchers were then able to validate their findings using long-read whole-genome sequencing. This highlights the important role of long-read whole-genome sequencing in identifying and reconstructing large-scale complex structural variants, particularly in DMD.

Implications From This Case Study

This case study demonstrates the careful, methodical approach needed to accurately detect and describe the pathogenic variants in DMD. The study authors concluded, “It is imperative to improve the detection rate and accurate reconstruction of pathogenic DMD variants for various reasons, including genetic counseling, prenatal diagnosis, and disease management in dystrophinopathies.”

Perhaps another question worth asking is whether such thorough investigations are warranted, especially in parts of the world where healthcare facilities are inadequate? Would it be more practical to simply assume a diagnosis of DMD based on key clinical features and to administer treatment accordingly? Although hardly a point of debate in the affluent West, detailed DNA and molecular analysis is often unavailable in some parts of the world or is only offered on a highly selective basis.

This explains the global asymmetry in the volume of DNA and molecular research, which is virtually absent from some parts of the world. Perhaps a more hopeful vision of medicine would be the equitable access to proper DNA testing when required as a guaranteed first step in the diagnosis and treatment of rare diseases such as DMD. 


Xie Z, Sun C, Zhang S, et al. Long-read whole-genome sequencing for the genetic diagnosis of dystrophinopathies. Ann Clin Transl Neurol. 2020;7(10):2041-2046. doi:10.1002/acn3.51201

Barseghyan H, Tang W, Wang RT, et al. Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis. Genome Med. 2017;9(1):90. doi:10.1186/s13073-017-0479-0