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#apaperaday: Diversity of mutations in the dystrophin gene and details of muscular lesions in porcine dystrophinopathies

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled:  Diversity of mutations in the dystrophin gene and details of muscular lesions in porcine dystrophinopathies

Today’s pick is on dystrophin variants in pigs from veterinary pathology by Kamiya et al. Origami figures to commemorate the Japanese authors. I cannot origami a pig yet, but can now also make a cat and a sloth. DOI: 10.1177/03009858231214028

In humans dystrophinopathies lead to muscle pathology and loss of muscle function. Pathogenic variants that result in the absence of dystrophin while those allowing production of partially functional dystrophins cause Becker. The dystrophin gene is located on the X-chromosome.

Females with pathogenic variants on one allele often do not have symptoms, but some do. This is called female dystrophinopathy and the symptoms can vary from mild to severely debilitating. Here authors look into pork muscle with dystrophic pathology.

Interestingly, they found these samples during meat inspections of pork meat. The inspectors noticed in some of the 6 months old pork muscle that there was fatty infiltrations. 28 muscle samples from 25 pigs were obtained for analysis.

For some frozen samples were available so also RNA and protein analysis could be performed. RNA analysis revealed 14 missense variants, 6 of which were thought to be pathogenic. Furthermore, 3 in-frame skipping events involving exon 35, 71 and 74 were observed.

All pigs showed exon 9 skipping both the ones with dystrophic pathology and normal pigs. Authors conclude this is likely alternative splicing. Note that also in humans exon 9 is spontaneously skipped in a subset of transcripts normally.

Histological analysis revealed fatty infiltration (see image below). his looks very similar to dystrophinopathy in humans. Authors also found inflammation and regeneration. When staining for dystrophin authors noticed reduced staining, especially for the C-terminal antibodies.

This suggests the pigs had a Becker-type dystrophinopathy rather than Duchenne, which makes sense because the lack of dystrophin causes very severe pathology in pigs and most do not survive the first few weeks. So they would not be accidentally found when butchered at 6 months.

Authors assume the loss of dystrophin staining is due to the missense and exon skipping variants. However, some of the missense variants are predicted to be benign – normally I would suggest they might cause missplicing. However, authors checked the mRNA!

It is possible that these variants do cause the pathology, but it is also possible that something else is going on, e.g. a sarcoglycanopathy can also lead to reduced dystrophin staining. Authors performed Western blotting as well, however, this is not that informative.

Authors did not include a protein reference control. This means lack of dystrophin can be due to true lack of dystrophin or because the protein lysate was not optimal (protein degradation due to e.g. freeze-thaw cycles or not freezing or storing the sample properly).

Authors discuss that 1 of the missense variants was independently found and reported in the USA suggesting that missense variants arise relatively frequently in the porcine dystrophin gene. For some pigs they found multiple variants.

Some of the pigs were females, where one would not expect dystrophinopathy. However, some of the variants were homozygous. Authors also discuss that splicing variants with cryptic splicing and exon skipping are more common in pigs than humans. However in humans often missplicing variants are not found, because only DNA is analyzed, and then usually only the exons. Here authors started the other way around, with muscle RNA. For humans that would involve a muscle biopsy.

Authors could not perform in-depth functional analyses on the pigs, as the muscles were inspected post-mortem. However, no overt functional problems were reported for the pigs, so the pathology was there in the muscle but it did not lead to obvious symptoms.

Authors discuss that female dystrophinopathy is occurring in humans and that from the pig samples it is clear that even heterozygous dystrophin variants can cause muscle pathology. However, for humans females are only diagnosed when they present with symptoms.

It is possible that females with a dystrophin pathogenic variant on one allele do also have muscle pathology when you would assess their skeletal muscle in depth. However, this is generally not done as biopsies are invasive. So only women presenting with symptoms are analyzed.

It is clear from the images at least in pigs you can have muscle pathology without overt functional symptoms at 6 months of age. Obvious disclaimer: maybe symptoms would come at a later age, pigs in the wild might do worse than pigs in captivity (food ad libitum and no predators) and of course maybe these pigs have another underlying cause for their pathology (it does appear to be dystrophin-glycoprotein complex related though). While it all may seem a bit morbid, I do like that approach authors have taken to study the cause of the pathology in these pigs

This can teach us about the situation in humans from a pathology perspective, from a ‘please also consider missplicing variants’ perspective and especially from the female dystrophinopathy perspective.