Author: worldduchenne

Today’s pick is one of my own papers by van Putten et al in FASEB journal doi 10.1096/fj.12-224170. Here we used the fact that women/females inactivate one of their X-chromosomes to study how much dystrophin is needed for survival and function.

Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double-knockout mice

X-inactivation also occurs in mice, normally in the 50:50 random pattern. However, there is a mouse model where the gene (Xist) coordinating X-inactivation has been altered. The chromosome with this altered Xist is more likely to be inactivated. This means that if you cross females with this Xist mutation with mdx males, the resulting female offspring will preferentially inactivate the X-chromosome with the Xist mutation and a functional dystrophin gene.

As the process is skewed but still random, this results in females expressing anywhere between 2 and 40% dystrophin in their muscles. We first did this in mdx mice, but they are only mildly affected. Here, we studied this in an mdx/utrophin knockout (dko) background, so we could also study impacts on survival.

In a first study we subjected mice to a 12 week functional test regime starting at 4 weeks of age. DKO mice (no dystrophin) generally had to be humanely killed before the age of 12 weeks. The dko/xist mice (having between 3-16% dystrophin in their muscle) survived longer. We subdivided the mice in those having <4% dystrophin and those having >4% dystrophin.

Even mice with <4% dystrophin had increased survival, lower CK and better function than dko, however mice with >4% dystrophin performed even better – though still less than wild type. We then performed a long term survival study. Notably in these studies we could only see the dystrophin levels after the animals had been sacrificed (a blinded experiment).

The 6 dko mice all died before 12 weeks, while those with <4% dystrophin survived longer. For those with 4-15% dystrophin 64% was alive at 10 months, while all mice with >15% were still alive at this point. These mice also resembled wild types in function and appearance.

6 mice were randomly selected for a very long survival study. The mice with 3% and 27% dystrophin died at 17 and 25 months due to muscular dystrophy. Other mice (>30% dystrophin) died due to age related causes >2 years old or a gall bladder problem (17 months).

Additional studies showed that dystrophin levels correlated with TIMP-1 serum levels (in mouse – we have not been able to translate this finding for patients). Also when looking at muscle histology we observed improvements at <4% dystrophin but more improvement at higher levels.

While this now may seem like old news (the paper is from 2013), it was one of the first papers showing that little bits of dystrophin already can go a long way and that it was likely not required to restore dystrophin at 20% to have a functional effect (as was the consensus at that time)

There are 3 limitations to this study however 1. The study had to use females, while Duchenne primarily occurs in men. 2. The mice had dystrophin from birth, while in patients restoration of dystrophin occurs later in life. 3. The study was in mice and not in human, so the levels may not translate directly.

Today’s pick for the Women and Duchenne theme of World Duchenne Awareness Day focuses on female caregivers (usually mothers) of Duchenne patients and their needs. The paper is by Peay at al from the Journal of Genetic Counseling. Doi 10.1007/s10897-017-0141-4. A very important topic as caregivers often forget to care for themselves.

Psychosocial Needs and Facilitators of Mothers Caring for Children with Duchenne/Becker Muscular Dystrophy

Notably, caregivers can provide better care when their social and psychological needs are met. As such, the standards of care for Duchenne also include a ‘needs’ assessment of caregivers. These needs range from social or psychological support to e.g. cope with anxiety to financial needs and facilitating time for self-care.

Authors mention that mothers with a son with Duchenne generally adjust well and also find their caregiver experience rewarding. Here authors did a study in the aspects of lived experience of caregivers of Duchenne and Becker patients. The goal was to identify psychosocial needs, respite needs and factors that facilitate caregiving.

Authors did an online survey with 205 caregivers, all biological mothers of Duchenne or Becker patients aged 27-71. 85% had a son with Duchenne, others with Becker or an intermediate phenotype. Patients were aged 1-40 years.

Over 50% of mothers indicated a medium or high need for psychosocial support to deal with uncertainty about the future, manage fears related to Duchenne or Becker and to cope with being a mother of a son with Duchenne.

70% indicated that their child depended on care (was not independent). 21% used respite care, while 17% indicated it was too much trouble and 57% indicated they felt uncomfortable leaving the care of their son to others, while 53% indicated their son would be willing to be cared by others. 69% indicated they needed a break occasionally.

Mothers also indicated that care facilitators were their partners and extended family. When asked about self-care most indicated they exercised or had a hobby. In the discussion authors stress that mothers tend to prioritize the worries for their own child over their own needs. This means caregivers need to proactively ask about the needs for psychosocial support.

Interestingly, the needs appear to be larger for mothers with younger children – though they require less care and support. With time mothers adjust more. Authors stress that caregivers should be aware of this and proactively offer support. They speculate that a diagnostic odyssey may play a role as well with the increased worry in mothers. Earlier diagnosis might be a way to reduce anxiety.

Mothers indicated that they learned a lot from their son’s approach to life. Authors discuss limitations of the study – they recruited from a database, so this does not cover all mothers. Also, mothers who are too busy with coping, would not be able to fill out the survey.

Authors indicate that more study is needed to allow optimal care provisions for mothers with sons with Duchenne and also to study the long-term care needs. Shout out to all the mothers of sons with Duchenne for the amazing work they do!

Photo by Gabe Pierce on Unsplash

Women and Duchenne theme continued with another paper trying to elucidate why some female carriers have symptoms and others have not. Today’s pick is from BMC medical genetics by Brioschi et al from Alessandra Ferlini’s group. Doi 10.1186/1471-2350-13-73

Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype

Authors introduce that it makes sense that some women have Duchenne, e.g. translocation mutations (see Sept 1 thread), or women with Turner syndrome and a dystrophin mutation (Turner patients have only 1 X-chromosome), or women who have two mutated dystrophin genes.

However, carriers have one functional and one mutated dystrophin gene. Authors wanted to elucidate why some carriers manifest symptoms, while others do not. They studied 7 manifesting and 11 non-manifesting carriers.

All 18 women had elevated CK levels, while the 7 manifesting carriers also had muscle weakness and pain with an age of onset of 2-43 years. None of the 18 women had heart pathology.

Staining of muscle biopsies showed a mosaic dystrophin pattern for both manifesting and non-manifesting carriers as well as signs of muscle damage and regeneration.

Authors then looked at X-inactivation patterns in the muscle biopsy. They used a proxy marker for this, the androgen gene (also located on the X-chromosome). In addition, they studied how much dystrophin was expressed from each of the two genes (so mutated and functional transcripts).

Interestingly, there was little correlation between X-inactivation and how much dystrophin was expressed from each gene. This suggests that skewed X-inactivation is not a likely cause of manifesting symptoms for carriers.

Authors note that for the 1 patient with the most severe symptoms, dystrophin levels were very low, while they were higher for an asymptomatic carrier. However, they performed this analysis only for these 2 individuals so it is premature to really draw strong conclusions.

It is good that authors looked at X-inactivation patters in muscle rather than blood (as is often done). However, the pattern in one piece of one muscle may still not be reflective of all the muscles. There is no way around this problem so authors did as well as they could.

Unfortunately, while the paper reveals that skewed X-inactivation is likely NOT to underly the manifesting of symptoms for patients, it does not reveal what IS the cause. Sadly, this is still unknown.