Carriers Of Muscular Dystrophy Research Articles

Loss of endogenous estrogen alters mitochondrial metabolism and muscle clock‐related protein Rbm20 in female mdx mice

AbstractFemale carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT‐labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates, and citrate synthase activity but that these deficits are offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA‐binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggest that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post‐menopausal DMD carriers.

Cardiovascular phenotype of the duchenne muscular dystrophy carrier female rat

Abstract Background Duchenne muscular dystrophy (DMD) is a lethal, severely progressive muscle-wasting disease. It is an X-linked recessive disease caused by mutations in the DMD gene encoding dystrophin protein and consequently the absence of dystrophin protein synthesis. Besides the skeletal muscle abnormalities, DMD patients are often suffering cardiovascular symptoms include cardiac fibrosis, dilated cardiomyopathy, arrhythmia, and congestive heart failure. Heterozygous female DMD carriers rarely acquire skeletal muscle symptoms, however the cardiac abnormalities are often underrepresented. There are few studies to date characterizing cardiovascular abnormalities in female DMD carriers and the mechanisms underlying the cardiac manifestations are still not fully known. Aims and Methods: Therefore, the aim of this study was to investigate the cardiovascular phenotype of 9 months old female DMD carrier rats and compare with age-matched wildtype rats. Objectives included assessment of cardiac function via transthoracic echocardiography, vascular function using wire myography, cardiac fibrosis and dystrophin via histology, mRNA expression of inflammatory markers via RT-qPCR, measurements of ACE1 and ACE2 activity, as well as expression of dystrophin, SERCA, sarcolipin, BAG3, and Connexin43 (Cx43) using western blotting. The cardiac function, vascular endothelial function, ACE1 and ACE2 activity, and collagen deposition in cardiac tissue were similar between the female DMD carrier and wildtype rats. The echocardiography data revealed thinning of the left-ventricular posterior wall indicating trend towards cardiac atrophy and dysfunction (p<0.05). Wire myography data showed trends of impaired endothelium-independent relaxation and hypercontractility, suggesting the potential vascular abnormalities. Histological analysis showed thickening of intima-media layer in coronary arteries, addition to reduced dystrophin staining, further affirming dystrophin loss impairs smooth muscle cell function (p<0.05, respectively). Protein expression of dystrophin, SERCA2, and BAG3 was decreased in female DMD carrier rats in the myocardium. We also found decreased levels of Cx43 at the intercalated discs, with a concomitant increase in the phosphorylation of Serine368 on Cx43 that very often precedes ubiquitination and degradation of Cx43 channels. In agreement, higher levels of the ubiquitin ligase Nedd4 were observed in hearts of female DMD carrier rats, implicating Cx43 degradation in cardiac dysfunction observed in these animals. Conclusions These findings indicate that female DMD carrier rats do not develop severe cardiac systolic dysfunction nor dilated cardiomyopathy at the age of 9 months unlike male DMD rats as previously described(1). However, the female DMD carrier rats begin to manifest tendencies towards cardiac and vascular dysfunction in association with a changes of calcium handling protein and Cx 43 expression in the heart.

Modeling Reduced Contractility and Stiffness Using iPSC-Derived Cardiomyocytes Generated From Female Becker Muscular Dystrophy Carrier

Study investigators encountered a female Becker muscular dystrophy (BMD) carrier with advanced heart failure (HF) and identified a stop-gain variant in procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) as a potential second-hit variant. Isogenic induced pluripotent stem cells (iPSCs) with dominant expression of WT-DMD, Δ45-48-DMD, or Δ45-48-DMD with corrected PLOD3 variant were established. Microforce testing using 3-dimensional self-organized tissue rings (SOTRs) generated from iPSC-derived cardiomyocytes (iPSC-CMs) demonstrated that correction of the heterozygous PLOD3 variant did not improve the reduced force, but it significantly recovered the reduced stiffness in Δ45-48-DMD SOTRs. Correction of the PLOD3 variant restored collagen synthesis in iPSC-CMs. Our findings revealed the pathogenesis underlying advanced HF in a female BMD carrier.

P.26 Motor and cognitive manifestations of young female carriers of Duchenne muscular dystrophy (DMD): a prospective natural history study

Clinical manifestations of Duchenne muscular dystrophy (DMD) have been well documented. However, less is known about the impact of DMD gene mutations for young female carriers. This cross-sectional study seeks to address the gap in understanding of the impact of carrier status on motor skill and cognitive functioning during formative years of development. We examined the motor abilities and neuropsychological functioning of genetically confirmed female DMD carriers in childhood and adolescence. Age appropriate functional and motor outcome assessments were administered including the Neuromuscular Gross Motor Outcome (GRO), 100-meter timed test (100m), Performance of Upper Limb (PUL), and others. Additionally, we assessed intelligence, executive functioning, visual motor integration, behavioral functioning and emotional regulation using standardized testing. To date, 8 subjects have been enrolled, average 11.7 years of age (range: 5–17 years) through the Nationwide Children's Hospital clinics. Gross motor performance was variable across the full cohort ranging between 71–100% of total score on the GRO, 10-95% of average predicted performance on the 100m, and 85-100% of total score on the PUL. Fine motor behavior and visual-motor integration tests indicate this cohort may experience a higher rate of motor coordination deficit and fine motor slowing, as evidenced by performance on norm-referenced assessments including the Grooved Pegboard Test (z-scores range: -0.75 to -5.61; N=7) and Beery Visual Motor Integration Scale (percentiles range: 0.7 – 63; N=7). Individual and group performance on motor outcome assessments and cognitive performance in areas related to attention, organization, working memory, arithmetic/mathematics, and verbal fluency to be presented. Subject enrollment, data collection, and analysis is ongoing.

A Paucisymptomatic HyperCKemia Patient Diagnosed with Manifesting Female Duchenne Muscular Dystrophy Carrier

A Paucisymptomatic HyperCKemia Patient Diagnosed with Manifesting Female Duchenne Muscular Dystrophy Carrier

Newborn screening for Duchenne muscular dystrophy-early detection and diagnostic algorithm for female carriers of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common pediatric-onset form of muscular dystrophy, occurring in 1 in 5,000 live male births. DMD is a multi-system disease resulting in muscle weakness with progressive deterioration of skeletal, heart, and smooth muscle, and learning disabilities. Pathogenic/likely pathogenic (P/LP) variants in the DMD gene, which encodes dystrophin protein, cause dystrophinopathy. All males with a P/LP variant in the X-linked DMD gene are expected to be affected. Two to 20% of female heterozygotes with a P/LP variant develop symptoms of dystrophinopathy ranging from mild muscle weakness to significant disability similar to Becker muscular dystrophy. Recently, with improvements in therapies and testing methodology, there is stronger evidence supporting newborn screening (NBS) for DMD for males and females because females may also develop symptoms. A consented pilot study to screen newborns for DMD was initiated in New York State (NYS) and conducted from 2019 to 2021. The identification of female carriers and the realization of the subsequent uncertainty of providers concerning follow-up during the pilot led to the development of algorithms for screening and diagnosis of carrier females, including both NBS and cascade molecular testing of family members.

EP463: Inconclusive Duchenne Muscular Dystrophy (DMD) carrier screening and atypical SNP-based NIPS sex chromosomes analysis suggest maternal sex chromosome abnormality

As of 2021, ACMG has expanded the recommended carrier screening panel to constitute 113 genes, now including the X-linked Duchenne Muscular Dystrophy (DMD) gene. Copy number variant (CNV) analysis by next-generation sequencing (NGS) of DMD yields inconclusive results for a small number of individuals due to the duplication/deletion of all DMD exons and adjacent regions on the X chromosome. In addition, a small number of SNP-based noninvasive prenatal screening (NIPS) tests yield no results for sex chromosomes due to a suspected maternal X chromosome abnormality.

Global longitudinal strain detects subtle left ventricular systolic dysfunction in Duchenne muscular dystrophy patients and carriers

BackgroundWith the continuous improvement of the respiratory care of Duchenne muscular dystrophy patients, cardiac manifestations (heart failure and arrhythmias) become the leading causes of morbidity and mortality. Early identification of cardiac muscle affection is crucial to start anti-failure drugs that reverse remodeling and improve prognosis. This study aimed to detect subtle cardiac changes in Duchenne muscular dystrophy patients and carriers using electrocardiography and echocardiography.ResultsThis study included genetically diagnosed Duchenne muscular dystrophy patients (28 males) and carriers (25 females) and compared them to healthy gender-matched control groups. All study participants underwent clinical assessment, 12-lead electrocardiography, and global longitudinal strain augmented echocardiography. In the current study, Duchenne muscular dystrophy patients had higher heart rates, smaller left ventricular internal diameters, left atrial diameter, lower ejection fraction, and worse left ventricular global longitudinal strain in comparison with the control group. The global longitudinal strain inversely correlated with the age of Duchenne muscular dystrophy patients. The number of exon mutations did not affect electrocardiography and echocardiographic findings. Exon mutations 45–47 and 51–54 were significantly associated with an ejection fraction less than 60%. Duchenne muscular dystrophy carriers had smaller left ventricular wall diameters, left ventricular end-diastolic diameter, left atrial diameter, and worse left ventricular global longitudinal strain in comparison with the control group.ConclusionsLeft ventricular global longitudinal strain could detect subtle left ventricular systolic dysfunction in Duchenne muscular dystrophy patients and carriers before the decline of left ventricular ejection fraction.

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Pathologically and Genetically Diagnosed Subclinical Symptomatic Duchenne Muscular Dystrophy Carrier: Broadened Spectrum of Clinical Phenotype

A 29-year-old female presented with an elevated level of serum creatine kinase without subjective weakness. Neurologic examination showed the subtle motor weakness of the right arm. Muscle biopsy showed dystrophic changes and a mosaic pattern of dystrophin expression. The diagnosis was confirmed by multiplex ligation-dependent probe amplification and whole-exome sequencing, revealing heterozygous deletion of exon 44 in the DMD gene. Here, we introduce a subclinical symptomatic Duchenne muscular dystrophy carrier, which broadens the clinical spectrum of phenotype.

Impact of estrogen deficiency on diaphragm and leg muscle contractile function in female mdx mice.

Female carriers of Duchenne muscular dystrophy (DMD) presenting with DMD symptomology similar to males with DMD, such as skeletal muscle weakness and cardiomyopathy, are termed manifesting carriers. There is phenotypic variability among manifesting carriers including the age of onset, which can range from the first to fourth decade of life. In females, estrogen levels typically begin to decline during the fourth decade of life and estrogen deficiency contributes to loss of muscle strength and recovery of strength following injury. Thus, we questioned whether the decline of estrogen impacts the development of DMD symptoms in females. To address this question, we studied 6–8 month-old homozygous mdx female mice randomly assigned to a sham or ovariectomy (OVX) surgical group. In vivo whole-body plethysmography assessed ventilatory function and diaphragm muscle strength was measured in vitro before and after fatigue. Anterior crural muscles were analyzed in vivo for contractile function, fatigue, and in response to eccentric contraction (ECC)-induced injury. For the latter, 50 maximal ECCs were performed by the anterior crural muscles to induce injury. Body mass, uterine mass, hypoxia-hypercapnia ventilatory response, and fatigue index were analyzed by a pooled unpaired t-test. A two-way ANOVA was used to analyze ventilatory measurements. Fatigue and ECC-injury recovery experiments were analyzed by a two-way repeated-measures ANOVA. Results show no differences between sham and OVX mdx mice in ventilatory function, strength, or recovery of strength after fatigue in the diaphragm muscle or anterior crural muscles (p ≥ 0.078). However, OVX mice had significantly greater eccentric torque loss and blunted recovery of strength after ECC-induced injury compared to sham mice (p ≤ 0.019). Although the results show that loss of estrogen has minimal impact on skeletal muscle contractile function in female mdx mice, a key finding suggests that estrogen is important in muscle recovery in female mdx mice after injury.

Linked-Read Whole Genome Sequencing Solves a Double DMD Gene Rearrangement.

Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16–29 in DMD gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a “non-carrier” status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1–15 and 30–34. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16–29 deletion, while the second allele showed a 1–34 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16–29 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.

Usefulness of copy number variant detection following monogenic disease exclusion in prenatal diagnosis

AimFamilies with an adverse history of monogenic disease focus on single‐gene diagnosis instead of low‐depth whole‐genome sequence, during subsequent pregnancies. The aim of this study was to assess the potential usefulness of low‐depth whole‐genome sequencing (copy number variant sequencing [CNV‐seq]) detection following monogenic disease exclusion in prenatal diagnosis.MethodsA total of 285 families with a history of monogenic disease (of 41 different types; eliminated during the current pregnancy) were recruited and retrospectively analyzed. Low‐depth whole‐genome sequencing (CNV‐Seq, Next‐Seq CN500 platform) was performed for all fetuses.ResultsThe CNV detection results of the 285 samples were as follows: one case of 18‐trisomy chimera (0.35%), one case of pathogenic 3q29 microdeletion syndrome CNV (0.35%), four cases of variant of uncertain significance (VUS) CNVs (1.40%), and four cases of Duchenne muscular dystrophy (DMD) carriers (1.40%); and the remaining samples were normal (96.15%). Of note, 2/285 (0.70%) samples still exhibited pathogenic abnormalities. All positive samples were followed up where the two cases of pathogenic abnormalities elected the pregnancy termination, while the four VUS cases and four DMD‐carrier cases were born healthy.ConclusionIn cases where prenatal fetal monogenic disease has been ruled out, CNV detection is still beneficial and should be performed to prevent missed pathogenic CNVs. However, the costs need to be balanced against benefits, and the research will need to assess other types of testing.

Combination of regional anaesthetic techniques in a Duchenne Muscular Dystrophy carrier undergoing mastectomy

Combination of regional anaesthetic techniques in a Duchenne Muscular Dystrophy carrier undergoing mastectomy

DMD – BIOMARKERS & OUTCOME MEASURES: P.132 Symptomatic female carrier of Duchenne muscular dystrophy with a novel single-base deletion mutation mimicking exon deletion in MLPA

Duchenne/Becker muscular dystrophy (DMD/BMD) is one of the most common muscle disorders characterized by an X-linked recessive inheritance pattern affecting male patients traditionally. Most DMD/BMD carriers remain asymptomatic, while only 2.5–10% of carriers are symptomatic and classified as symptomatic carriers. A 50-year-old female patient visited the neurological outpatient clinic complaining of progressive weakness of the lower legs since childhood. She also complained of frequent falls while walking and presented with an enlarged calf and Gowers' sign.

Population-Wide Duchenne Muscular Dystrophy Carrier Detection by CK and Molecular Testing.

Carrier screening of Duchenne muscular dystrophy (DMD) has not been widely evaluated. To identify definite DMD female carriers prior to or in early pregnancy, we studied a large population of reproductive age females and provided informed reproductive options to DMD carriers. 37268 females were recruited from the Hangzhou Family Planning Publicity and Technology Guidance Station/Hangzhou Health Service Center for Children and Women, Hangzhou, China, between October 10, 2017, and December 16, 2018. CK activity was measured with follow-up serum DMD genetic testing in subjects with hyperCKemia, defined as CK > 200 U/L. The calculated upper reference limit (97.5th percentile) of serum creatine kinase (CK) for females aged 20-50 years in this study was near the reference limit recommended by the manufacturer (200 U/L), above which was defined as hyperCKemia. 427 females (1.2%) harbored initially elevated CK, among which 281 females (response rate of 65.8%) accepted CK retesting. DMD genetic testing was conducted on 62 subjects with sustained serum CK > 200 U/L and 16 females with a family history of DMD. Finally, 6 subjects were confirmed to be DMD definite carriers. The estimated DMD female carrier rate in this study was 1 : 4088 (adjusting for response rate), an underestimated rate, since only 50% to 70% of DMD female carriers manifest elevated serum CK, and carriers in this study may have been missed due to lack of follow-up or inability to detect all DMD pathogenic variants by current genetic testing.

Myocardial T1-mapping and Extracellular Volume Quantification in Patients and Putative Carriers of Muscular Dystrophy: Early Experience.

To assess myocardial fibrosis associated with muscular dystrophy, T1-mapping and extracellular volume fraction (ECV) quantification was prospectively performed using cardiovascular MR (CMR) imaging in 6 male patients with muscular dystrophy and 5 female putative carriers of Duchenne or Becker muscular dystrophy. Five patients and all putative carriers had an elevated ECV (>29.5% for men and >35.2% for women), suggesting that ECV has a potential to detect diffuse fibrotic changes in patients and putative carriers of muscular dystrophy.

Enhanced dimethylarginine degradation improves coronary flow reserve and exercise tolerance in Duchenne muscular dystrophy carrier mice.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac β-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.

Duchenne and Becker muscular dystrophy carriers: Evidence of cardiomyopathy by exercise and cardiac MRI testing

BackgroundVaried detection methods have resulted in conflicting reports on the prevalence of cardiac disease in Duchenne and Becker muscular dystrophy carriers (MDC). MethodsWe performed a prospective cohort study of 77 genetically-confirmed MDC mothers, 22 non-carrier mothers, and 25 controls. All participants underwent Cardiopulmonary Exercise Testing (CPET) and Cardiac Magnetic Resonance imaging (CMR). Results25% of carriers had ventricular ectopy in recovery of exercise (RecVE) as compared to 1 non-carrier and no controls (p = .003). No difference in age or maximal oxygen consumption was noted. 11 carriers had abnormal (<55%) left ventricular ejection fraction by CMR. Evidence of late gadolinium enhancement (LGE) was noted in 48% of MDC, 1 non-carrier patient and no control subjects (p < .0001). Subset analysis of LGE+ and LGE- subjects revealed differences in age (44.1 v 38.6 yrs.; p = .005), presence of RecVE, (38.9% v 10.5%, p = .004), and high serum creatine kinase (CK) (> 289 U/l; 52.8% v 31.6%, p = .065). ConclusionWe describe the prevalence of disease using CPET and CMR in genetically-proven MDC. 49% of carriers had fibrosis, opposed to 5% of non-carriers, highlighting the importance of genetic testing in this population. Despite cardiomyopathy, functional assessment by treadmill was normal, illustrating the discrepancy in cardiac and skeletal muscle impacts. Age, RecVE and serum CK appear to have an important role in predicting cardiomyopathy. Serum CK levels suggest that a systemic higher global disease severity and not tissue heterogeneity may be the etiology for greater cardiac disease and relatively spared skeletal muscle disease in this population.Clinical Trial Registrationhttps://clinicaltrials.gov/ct2/show/NCT02972580?term=mendell&cond=Duchenne+Muscular+Dystrophy&rank=5; ClinicalTrials.gov Identifier: NCT02972580

Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle.

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.