Follow-up Study on Female Carriers With DMD Gene Variants

Duchenne and Becker muscular dystrophies are X-linked recessive allelic disorders caused by mutations of the dystrophin gene on chromosome Xp21. The gene mutation causes the absence or very severe reduction of dystrophin protein in the muscle cells, triggering chronic myofiber damage, inflammation, and loss of muscle fibers. Muscle tissue is replaced by fibrous and adipose tissue which further leads to necrosis, progressive muscle weakness, and loss of independent ambulation \[1\].

Duchenne muscular dystrophy (DMD) is one of the commonest inherited disorders of muscle. Based on a systematic review of worldwide population-based studies the pooled prevalence of DMD and BMD was 4.78 (95% CI 1.94-11.81) and 1.53 (95% CI 0.26-8.94) per 100,000 males respectively \[2\].

The disorders preferentially affect males due to the X-linked inheritance. Female carriers may pass on the pathogenic variant to their daughters, resulting in a significant number of female carriers of pathogenic DMD variants. A third of all new cases are caused by de novo pathogenic variants \[3\].

Even though female carriers have one healthy X-chromosome, they are not necessarily asymptomatic as both muscular and cardiac involvement has been reported in carriers \[4-12\]. These females are classified as "manifesting carriers" \[13,14\]. The incidence of skeletal muscle involvement among female carriers of DMD was 2.5%-19%, and of dilated cardiomyopathy (DCM) 7.3%-16.7% for DMD carriers and 0%-13.3% for BMD carriers \[2\], but in one of the latest cross-sectional studies with some of the most sensitive outcome measures to date 81 % showed muscle affection \[4\] and 62 % cardiac dysfunction \[15\]. Since then, cardiac MRI techniques have been further developed, why the numbers might be even higher. Patients with BMD have not even been investigated with these techniques. Cardiomyopathy in female DMD and BMD carriers can be clinically significant. Therefore, adult unaffected dystrophinopathy carriers are recommended to undergo echocardiography every 5 years according to the clinical guidelines in Europe and the United States \[16,17\]. Carriers with cardiac affection are often examined even more frequently. However, no one has yet investigated the rate of progression, which can make it difficult to determine the frequency of clinical visits.

In the above-mentioned studies, there was a large variability in the severity of symptoms with some being asymptomatic and some having severe symptoms. Skewed X-Chromosome Inactivation (XCI) might explain some of this variability. When the X chromosome carrying the normal DMD gene is preferentially inactivated this will in theory lead to moderate-severe muscle involvement.

Some studies have observed that DMD carriers with moderate/severe muscle involvement, exhibit a moderate or extremely skewed XCI, in particular, if presenting with an early onset of symptoms, while DMD carriers with mild muscle involvement present a random XCI \[4, 18\]. However, former studies have generally had a low power, investigated the XCI in blood and not muscle, and have often not investigated asymptomatic vs symptomatic patients. Some studies are inconclusive and some even contradictory and therefore no conclusions can be made \[18\]. Thus, the underlying cause of the large variability in phenotype is therefore uncertain.

2.2 Aim

The aims of this study are thus:

1. To describe the change over a 6-year follow-up period in the structure and function of the heart and in function and muscle fat fraction in skeletal muscle of DMD/BMD carriers. 2. To explain the relationship between the XCI and the severity of the disease (phenotype). 3. To compare cardiac affection of female carriers of DMD/BMD to patients with BMD using new cardiac MRI techniques (spectroscopy and Dixon sequences).

3\. Methods 3.1 Study methods

This study contains three parts:

Part 1 is a 6-year follow-up on 53 genetically verified female carriers of pathogenic DMD variants initially investigated in 2016-2018 at Copenhagen Neuromuscular Center, Rigshospitalet (Ethical journal no. H-16035677). In this part, the same 53 females will be investigated with the same measurements as 6 years ago to describe the progression of symptoms. All the follow-up results from this study will be compared to the results from 6 years ago.

In Part 2 a muscle biopsy will be taken from 1-3 muscles (see "3.3.3 Description of outcomes) to investigate the XCI. To correlate the XCI to the phenotype, these patients will also undergo a muscle MRI and a Medical Research Council scale score for muscle strength (MRC).

In Part 3 The cardiac structure and function in patients with BMD will be investigated using a cardiac MRI to compare the findings with that of female carriers. An MRC will carried out to investigate if the heart affection correlates to the muscle affection.

Female carriers can decide whether to participate in Part 1, Part 2, or both. Patient with BMD can only participate in Part 3.

• Change in contractility [Time frame: 1 hour]
• Change in blood concentrations [Time frame: 1 hour]
• Change in blood concentrations [Time frame: 1 hour]
• Change in blood concentrations [Time frame: 1 hour]
• Change in blood concentrations [Time frame: 1 hour]
• Change in blood concentrations [Time frame: 1 hour]
• Change in lower extremity strength [Time frame: 1 hour]
• Change in questionnaires on fatigue [Time frame: 5 minutes]
• Progression of electrocardiographic findings [Time frame: 10 minutes]
• Correlation between cardiac structure and function [Time frame: 1 hour]