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#apaperaday: Comprehensive analysis of genomic complexity in the 5’ end coding region of the DMD gene in patients of exons 1–2 duplications based on long-read sequencing

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Comprehensive analysis of genomic complexity in the 5’ end coding region of the DMD gene in patients of exons 1–2 duplications based on long-read sequencing

Today’s pick is from Shen et al in @BioMedCentral Genomics on duplications in the beginning of the DMD encoding dystrophin gene with long read sequence analysis. DOI: 10.1186/s12864-024-10224-2

Dystrophinopathies are caused by pathogenic variants in the DMD gene. In Duchenne patients symptoms arise early and disease progresses quickly, while in Becker patients symptoms arise (slightly) later and disease progresses slower.

Authors here focus on duplications of one or more exons, which are found in ~10% of dystrophinopathies. When detecting these variants with MLPA (multiplex ligation-dependent probe amplification) you know which exons are duplicated but not where they are located.

For duplications this is important. For deletions, when they are gone, you know they are gone from the DMD gene. However, the duplicated exons can be within the DMD gene (disrupting the gene code) but they can also be elsewhere, e.g. before the gene or on another chromosome.

if that happens, the DMD gene can function normally. If a patient presents with dystrophinopathy symptoms & duplications are found, it is safe to assume they are in the DMD gene. However, when screening is used in e.g. individuals who want to become pregnant, this is not clear.

Here authors present 3 families with duplications in the beginning of the DMD gene. The first family had a duplication of exon 1-2 that was identified during screening in a healthy female. 3 female and 3 male relatives had the same duplication, suggesting it was not pathogenic.

Long read sequencing revealed the duplication was located before the DMD gene, so normal transcripts could be produced. This is not the first time this duplication is reported as not pathogenic, see e.g. collaborative work I was involved in.

The second family had a patient with Duchenne symptoms and an exon 2 duplication. This is out-of-frame so you expect Duchenne. His sister had female dystrophinopathy with elevated blood damage marker, while the mother was a carrier without symptoms.

Long read sequencing confirmed the exon 2 duplication was within the DMD gene. The 3rd family had a complex duplication of cortical dystrophin exon 1 (Dp427c). You would expect this NOT to cause Duchenne, as this variant would be expected to leave the muscle transcript intact.

However, the family showed Duchenne like pathology. Long read sequencing revealed that the Dp427c was duplicated, but that there was more going on: an inversion and the duplication was not before Dp427m (as expected, because Dp427c is before Dp427m) but between Dp427m & exon 2.

Authors predict this will cause an out of frame transcript. Because the Dp427c duplicated exon is inverted, I do not think it will be spliced between Dp427m and exon 2, so in theory this should be in-frame. However, this duplication makes the first intron very long.

So it is likely that splicing is not happening properly anymore (intron 1 for Dp427m is normally already 120 kb, with probably an extra 100 kb things might go wrong). Authors discuss they did not have muscle tissue from this family so they cannot assess mRNA.

As such we can only speculate about the mechanism. This analysis does make clear however that we should not immediately classify exon 1 duplications as benign. They have the potential to be pathogenic. The best analysis is to assess male family members.

If these have the duplication but do not have muscle symptoms then you can conclude it is benign. Alternatively, long read sequencing can also allow identification of where the duplication is located, but as outlined by the Dp427c case, even then mRNA effects are not always clear

Authors stress that discerning whether a patient has Duchenne or Becker is sometimes challenging. I agree, the field is moving towards dystrophinopathy more and more as with good care, things are not longer black and white (ambulant >16 yrs Becker, wheelchair <12 yrs Duchenne)

Even before steroids & care standards, there were ‘intermediate’ patients who did not fit the Becker & Duchenne criteria. Dystrophinpathy also allows easier inclusion of female dystrophinopathy: manifesting carrier sounds benign, while some females have debilitating symptoms.

Back to the paper: nice work and good to keep mentioning that the exon 1-2 duplications are likely benign but that the DMD gene will always have some surprises and exceptions.