Category: News

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.

Today and tomorrow we dive into the cause of manifesting carriers starting with a paper by Soltanzadeh et al in Neuromuscular Disorders. Doi 10.1016/j.nmd.2010.05.010

Clinical and genetic characterization of manifesting carriers of DMD mutations

While most women who have a mutation in one of their dystrophin genes do not have symptoms, 2-8% does present with symptoms. The severity of these symptoms varies and they involve mainly muscle weakness and pain. For some manifesting carriers, the severity can be almost as severe as Duchenne.

Two days ago I explained about X-inactivation: when the embryo is in a ~100 cell stage either the X-chromosome of the mother or that of the father is randomly inactivated in each of the cells and that inactivation pattern is then inherited by all daughter cells.

This means that carriers will have ~50% of muscle fibers that express dystrophin (mutated dystrophin gene inactive) and 50% of muscle fibers that do not express dystrophin (functional dystrophin gene inactive).

That is indeed what one sees when a biopsy of a female carrier is stained for dystrophin. Furthermore, some pathology is generally also visible: variation in muscle fiber size, centrally located nuclei (sign of recent regeneration) and damage/necrosis and inflammation.

In this paper authors studied how often manifesting carriers occurred in their database. Out of 860 female subjects, 15 manifesting carriers were identified (weakness and myalgia). 8 had a relative with Duchenne, while 7 did not. Of the 15, 5 had dilated cardiomyopathy (DCM).

Yesterday I stressed that carriers are at risk for developing DCM in the absence of muscle symptoms. When carriers manifest muscle symptoms, the risk of also developing DCM increases.

Authors analyzed biopsies of some of the manifesting carriers and confirmed they had the pathological signs expected. Also the mosaic dystrophin staining pattern was observed.

Authors looked into reasons why these carriers manifested symptoms. For 1 the reason was clear: she carried two mutations in the dystrophin gene – likely one on each of her dystrophin genes. This was a woman who was quite severely affected, as would be expected.

For the other carriers authors speculated X-inactivation might have been skewed. Because the process is random it is possible that instead of 50:50, an 80:20 pattern occurs (or 70:30 etc). When the functional gene is inactivated with a higher frequency than the mutated gene, less dystrophin can be produced is the hypothesis.

Authors looked at X-inactivation in the blood cells of the carriers and found that for 5 manifesting carriers, X-inactivation was skewed while for others it was not. Note that it is possible the X-inactivation patters between muscle and blood cells varies, so their results may not have been predictive for muscle in all cases.

The most important lessons from this paper are that dilated cardiomyopathy is frequent in manifesting carriers. Clinicians should be aware of this. Secondly, women can be a carrier of the dystrophin mutation and manifest symptoms also if they do not have a relative with Duchenne.

In fact, about 50% of the cases discussed here did not have a relative with Duchenne. This resulted in delays in identifying the cause of the symptoms. In fact, one of the women underwent a liver biopsy because her transaminase enzymes were elevated.

These enzymes occur in liver but also in muscle and will leak into blood upon damage of either.

This paper underlines that awareness of women with dystrophin mutation being at risk for heart and muscle pathology is important to initiate genetic counseling and but also to prevent incorrect medical intervention and allow proper medical intervention.

Today’s pick in #apaperaday “Women and Duchenne” special is a letter to the editor by Wang et al in the journal of cardiac failure. 10.1016/j.cardfail.2022.03.359 The letter is short but makes an important point. It reflects on an earlier publication in the journal on the importance of cardiac care and monitoring for Duchenne patients.

A Parallel Need for Cardiovascular Care for Female Carriers of Duchenne and Becker Muscular Dystrophy

Authors here stress to ALSO include female carriers in the monitoring practice. 2/3 mothers with a Duchenne son will be a carrier. Carriers of the dystrophin mutation have 7-16% chance of developing dilated cardiomyopathy. Research has shown that up to 48% of female carriers have fibrosis in their hearts.

Guidelines prescribe checking for heart function in carriers starting in adulthood. This involves a physical examination, ECG and imaging every 3-5 years and more frequent if results dictated this.

Authors stress however that this does not happen in reality. In their institute (Penn Medicine Center) they have now launched a dedicated clinic to focus on long term care and monitoring of female carriers.

As the #WDAD2022 theme is Women and Duchenne, I thought it was important to stress this point. Two out of three mothers with a son with Duchenne is at risk of developing dilated cardiomyopathy. Once this is identified, action can be taken (intervention with drugs and careful monitoring).

Today we kick off with a paper from the Journal of Medical Genetics by Lindenbaum et al from 1979 on a female with Duchenne. Since the dystrophin gene, which is mutated in Duchenne is located on the X-chromosome, Duchenne occurs primarily in males. DOI 10.1136/jmg.16.5.389

Muscular dystrophy in an X; 1 translocation female suggests that Duchenne locus is on X chromosome short arm

First some context: Females have 2 X-chromosomes so they have a back-up copy, while men have only 1 X-chromosome so if their dystrophin gene is not functional, they have no dystrophin. In 1979 the dystrophin gene (formally called DMD gene) was not yet identified.

Researchers knew it had to be on the X-chromosome but did not know where. This made genetic counseling very difficult, so researchers were ‘hunting’ for the location of the gene. Notably, females with Duchenne with a specific mutation type (translocation) have been crucial for finding the location!

A translocation is when part of one chromosome is exchanged with part of another chromosome (say chromosome 1). When this occurs with the X-chromosome, the ‘break’ can go through the dystrophin gene. Now part of the gene is on chromosome 1 and part is on chromosome X. This disrupts the code because it cannot longer be produced from start to end.

However, the other X-chromosome of the woman still has the functional dystrophin gene. Why is that not used to produce dystrophin? This is due to a process called X-inactivation. When a female embryo is ~200 cells, each cell will inactivate one of its 2 X-chromosomes randomly. That pattern is then inherited by all daughter cells.

Generally, this occurs with 50% of cells inactivating the X-chromosome inherited from the father and 50% inactivating the X-chromosome inherited from the mother. However, when a translocation occurs, the X-chromosome that is combined with part of chromosome 1 will inactivate also part of chromosome 1.

This is not compatible with life. So, the cells in which that happens will die and only the cells where the non-translocated X-chromosome is inactivated will survive. This means that this female embryo will not be able to produce dystrophin. The X-chromosome with the functional dystrophin gene is not active.

Now to the paper: Authors report a girl who at 5 years of age showed all the signs and symptoms of Duchenne:

• Hypertrophic calves
• High CK
• Difficulty with walking and stair climbing
• Gowers maneuver

The girl required a wheelchair at age 8 (this was before steroids were used).

A biopsy was done and the analysis revealed the typical features of Duchenne (inflammation, regeneration, fibrosis). Note that this was before the gene and the protein were found, so authors could not check whether dystrophin was present or absent and had to rely on symptoms and histology only.

Chromosomal analysis revealed a translocation between chromosome 1 and chromosome X. Due to finding the location of this translocation breakpoint, and those of 2 other females with translocations and Duchenne, researchers could zoom in on the location of the dystrophin gene on Xp21

This later resulted in the identification of the gene in 1986 and the dystrophin protein in 1987. Already in 1985 genetic counseling could be offered to some families using the regions known to be part of the dystrophin gene identified with this ‘gene mapping’ technique.

So, women with Duchenne have played a crucial role in finding the dystrophin gene as back then the techniques could not pick up the deletions within the gene that occur in most men with Duchenne.