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Population-based incidence and prevalence of facioscapulohumeral dystrophy
Abstract
Objective:
To determine the incidence and prevalence of facioscapulohumeral muscular dystrophy (FSHD) in the Netherlands.
Methods:
Using 3-source capture-recapture methodology, we estimated the total yearly number of newly found symptomatic individuals with FSHD, including those not registered in any of the 3 sources. To this end, symptomatic individuals with FSHD were available from 3 large population-based registries in the Netherlands if diagnosed within a 10-year period (January 1, 2001 to December 31, 2010). Multiplication of the incidence and disease duration delivered the prevalence estimate.
Results:
On average, 52 people are newly diagnosed with FSHD every year. This results in an incidence rate of 0.3/100,000 person-years in the Netherlands. The prevalence rate was 12/100,000, equivalent to 2,000 affected individuals.
Conclusions:
We present population-based incidence and prevalence estimates regarding symptomatic individuals with FSHD, including an estimation of the number of symptomatic individuals not present in any of the 3 used registries. This study shows that the total number of symptomatic persons with FSHD in the population may well be underestimated and a considerable number of affected individuals remain undiagnosed. This suggests that FSHD is one of the most prevalent neuromuscular disorders.
... The prevalence of FSHD was estimated at 12/100 000 using capture-recapture methodology in the Netherlands. 9 This is higher than estimates of 5/100 000 prior to genetic testing. 9 Considerable numbers of clinically affected individuals remain genetically undiagnosed, suggesting that the prevalence rate of 12/100 000 is conservative. ...
... 9 This is higher than estimates of 5/100 000 prior to genetic testing. 9 Considerable numbers of clinically affected individuals remain genetically undiagnosed, suggesting that the prevalence rate of 12/100 000 is conservative. 9 FSHD is caused by derepression of the embryogenic transcription factor DUX4 in muscle cells, the expression of which is hypothesized to be toxic, leading to cell death. ...
... 9 Considerable numbers of clinically affected individuals remain genetically undiagnosed, suggesting that the prevalence rate of 12/100 000 is conservative. 9 FSHD is caused by derepression of the embryogenic transcription factor DUX4 in muscle cells, the expression of which is hypothesized to be toxic, leading to cell death. The DUX4 gene is located at the distal end of the macrosatellite repeat array D4Z4 in the subtelomeric region on chromosome 4q (sub-band 4q35.2). ...
The gold standard for facioscapulohumeral muscular dystrophy (FSHD) genetic diagnostic procedures was published in 2012. With the increasing complexity of the genetics of FSHD1 and 2, the increase of genetic testing centers, and the start of clinical trials for FSHD, it is crucial to provide an update on our knowledge of the genetic features of the FSHD loci and renew the international consensus on the molecular testing recommendations. To this end, members of the FSHD European Trial Network summarized the evidence presented during the 2022 ENMC meeting on Genetic diagnosis, clinical outcome measures, and biomarkers. The working group additionally invited genetic and clinical experts from the USA, India, Japan, Australia, South‐Africa, and Brazil to provide a global perspective. Six virtual meetings were organized to reach consensus on the minimal requirements for genetic confirmation of FSHD1 and FSHD2. Here, we present the clinical and genetic features of FSHD, specific features of FSHD1 and FSHD2, pros and cons of established and new technologies (Southern blot in combination with either linear or pulsed‐field gel electrophoresis, molecular combing, optical genome mapping, FSHD2 methylation analysis and FSHD2 genotyping), the possibilities and challenges of prenatal testing, including pre‐implantation genetic testing, and the minimal requirements and recommendations for genetic confirmation of FSHD1 and FSHD2. This consensus is expected to contribute to current clinical management and trial‐readiness for FSHD.
... Aerobic rehabilitation programs and combined programs including aerobic exercise and moderate resistance training can be safely used in subjects with neuromuscular diseases, particularly FSHD [7,8]. As Voorn et al. [9] pointed out, protocols need to be personalized, their adaptation to each individual's capacity requiring an initial test and not just the prediction of one's capacity to increase adherence and effectiveness of protocols. ...
... Aerobic performance was evaluated by measuring VO2 max and maximum aerobic power (MAP) during a stress test on an ergocycle. Walking performance was assessed using a 6-minute walk test and a 10-m walk test (10mWT) [7]. Muscle strength assessments were performed on the knee flexors/extensors of both lower limbs using the isokinetic test (in concentric mode) [7]. ...
... Walking performance was assessed using a 6-minute walk test and a 10-m walk test (10mWT) [7]. Muscle strength assessments were performed on the knee flexors/extensors of both lower limbs using the isokinetic test (in concentric mode) [7]. To investigate sleep, we used the Insomnia Severity Index (ISI) [14] and a visual number scale to measure physical fatigue. ...
Facioscapulohumeral muscular dystrophy (FSHD) is a common form of adult muscular dystrophy often resulting also in cardiorespiratory deconditioning and weakness of the lower limbs. Although previous studies examined outcomes of interventions aimed at improving either cardiorespiratory fitness or muscle strength, the potential benefits of a rehabilitation program targeting both remain unexplored. Thus, the aim of this study was to evaluate changes following participation in a rehabilitation program combining aerobic and strength exercises. We conducted a retrospective analysis of 10 subjects with FSHD who participated in our rehabilitation program during 2018 and 2019. Each of the 20 sessions consisted of aerobic training on a cycloergometer and a moderate lower limb strength exercises on an isokinetic machine in combination with conventional therapy. The primary outcomes were walking speed, aerobic performance and isokinetic strength of the knee extensors and flexors. The secondary outcomes were fatigue, insomnia. VO2max and walking speed increased significantly by 2.125 ml·kg ⁻¹ ·min ⁻¹ [95% confidence interval (CI): 0.75–3.62, P = 0.022] and 0.28 m/s (95% CI: 0.16–0.4, P = 0.002), respectively. The effect size was small for V02max (Hedge’s g, 0.44; 95% CI: −0.5 to 1.37) and large for walking speed (Hedge’s g, 0.99; 95% CI: 0.06–1.92). The knee flexor strength significantly increased at rehabilitation discharge (repeated measures analysis of variance P = 0.004). Positive changes in fatigue and insomnia were also observed. Our preliminary results provide evidence that a relatively short course of a comprehensive rehabilitation program targeting both cardiorespiratory fitness and knee muscle strength can be beneficial for people with FSHD, which warrants further prospective studies.
... Facioscapulohumeral dystrophy (FSHD) is the third most prevalent muscular dystrophy in adults affecting between 1/8000-1/20 000 individuals world-wide [1]. Genetic and molecular biology studies determined that FSHD is caused by the aberrant expression of the transcription factor DUX4 in skeletal muscle [2][3][4]. ...
... Here, our goal is to curate a subset of DUX4-targeted genes to represent the DUX4 signature with better performance in discriminating mild-to-moderately affected muscles on molecular levels. The workf low (Supplementary Fig. 2a) started with 67 DUX4-targeted genes that are robustly up-regulated in DUX4target-positive FSHD biopsy samples, FSHD myotubes, and DUX4transduced muscle cells [25], followed by [1] lifting the coordinates and annotation from the initially used hg19 to GRCh38 genome build; [2] removing duplicated or deleted DUX4 targeted genes in the GRCh38 genome build instead of previously used hg19; and [3] removing four TRIM 49 variants whose gene expressions were synchronized with TRIM49; the same applied to three TRIM53 variants and two TRIM43 variants. ...
Identifying the aberrant expression of DUX4 in skeletal muscle as the cause of facioscapulohumeral dystrophy (FSHD) has led to rational therapeutic development and clinical trials. Several studies support the use of MRI characteristics and the expression of DUX4-regulated genes in muscle biopsies as biomarkers of FSHD disease activity and progression. We performed lower-extremity MRI and muscle biopsies in the mid-portion of the tibialis anterior (TA) muscles bilaterally in FSHD subjects and validated our prior reports of the strong association between MRI characteristics and expression of genes regulated by DUX4 and other gene categories associated with FSHD disease activity. We further show that measurements of normalized fat content in the entire TA muscle strongly predict molecular signatures in the mid-portion of the TA, indicating that regional biopsies can accurately measure progression in the whole muscle and providing a strong basis for inclusion of MRI and molecular biomarkers in clinical trial design. An unanticipated finding was the strong correlations of molecular signatures in the bilateral comparisons, including markers of B-cells and other immune cell populations, suggesting that a systemic immune cell infiltration of skeletal muscle might have a role in disease progression.
... Facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant disorder, is one of the most common muscular dystrophy in adults in Europe [1][2][3]. It is initially characterized by weakness and atrophy of face and scapular muscles [4]. ...
... Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder characterized by progressive and asymmetric weakening of facial, scapular girdle, and upper limb skeletal muscles [1,2]. It is one of the most prevalent disorders of muscular dystrophy with a prevalence of 1:20,000 to 1:8,000 [3,4]. FSHD has been categorized into two subtypes [5]. ...
Background
Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow.
Methods
We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case–control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES).
Results
Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES.
Conclusions
Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.
... Later in the disease course, lower limb and trunk muscles become affected [1,2]. It is the second most common inherited muscular dystrophy in adulthood, with an estimated prevalence of 1 per 8,000 to 1 per 20,000 [3,4]. FSHD type 1 (95% of all patients [5]) is caused by contraction of D4Z4 microsatellite repeats on chromosome 4. ...
Background: Facial weakness is a key feature of facioscapulohumeral muscular dystrophy (FSHD) and may lead to altered facial expression and subsequent psychosocial impairment. There is no cure and supportive treatments focus on optimizing physical fitness and compensation of functional disabilities. Objective: We hypothesize that symptomatic treatment options and psychosocial interventions for other neurological diseases with altered facial expression could be applicable to FSHD. Therefore, the aim of this review is to collect symptomatic treatment approaches that target facial muscle function and psychosocial interventions in various neurological diseases with altered facial expression in order to discuss the applicability to FSHD. Methods: A systematic search was performed. Selected studies had to include FSHD, Bell’s palsy, Moebius syndrome, myotonic dystrophy type 1, or Parkinson’s disease and treatment options which target altered facial expression. Data was extracted for study and patients’ characteristics, outcome assessment tools, treatment, outcome of facial expression and or psychosocial functioning. Results: Forty studies met the inclusion criteria, of which only three studies included FSHD patients exclusively. Most, twenty-one, studies were performed in patients with Bell’s palsy. Studies included twelve different therapy categories and results were assessed with different outcomes measures. Conclusions: Five therapy categories were considered applicable to FSHD: training of (non-verbal) communication compensation strategies, speech training, physical therapy, conference attendance, and smile restoration surgery. Further research is needed to establish the effect of these therapies in FSHD. We recommend to include outcome measures in these studies that cover at least cosmetic, functional, communication, and quality of life domains.
... FSHD causes progressive wasting of facial, shoulder, and upper arm as well as lower leg musculature. 1,2 The majority of FSHD cases (>95%) are caused by monoallelic contraction of 3.3 kb D4Z4 macrosatellite repeat sequences located at the subtelomeric region of chromosome 4q (4qter D4Z4) (FSHD1 (MIM 158900)). 3,4 FSHD1 is associated with 1$10 copies of D4Z4 repeats in the contracted allele in contrast to 11-150 copies in the intact allele. ...
Facioscapulohumeral dystrophy (FSHD) is linked to contraction of D4Z4 repeats on chromosome 4q with SMCHD1 mutations acting as a disease modifier. D4Z4 heterochromatin disruption and abnormal upregulation of the transcription factor DUX4, encoded in the D4Z4 repeat, are the hallmarks of FSHD. However, defining the precise effect of D4Z4 contraction has been difficult because D4Z4 repeats are primate-specific and DUX4 expression is very rare in highly heterogeneous patient myocytes. We generated isogenic mutant cell lines harboring D4Z4 and/or SMCHD1 mutations in a healthy human skeletal myoblast line. We found that the mutations affect D4Z4 heterochromatin differently, and that SMCHD1 mutation or disruption of DNA methylation stabilizes otherwise variegated DUX4 target activation in D4Z4 contraction mutant cells, demonstrating the critical role of modifiers. Our study revealed amplification of the DUX4 signal through downstream targets, H3.X/Y and LEUTX. Our results provide important insights into how rare DUX4 expression leads to FSHD pathogenesis.
... The severity of the disease varies with age and sex. Thus, the degree of muscle damage shows a vast difference at the clinical level, ranging from almost asymptomatic weakness related to eye closure to significant disability with weakness of the shoulder and pelvic girdle and bilateral leg drop (1,2). These clinical manifestations are characteristic of the highly heterogeneous nature of the FSHD phenotype. ...
Objective
Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is one of the most common forms of autosomal-dominant muscular dystrophies characterized by variable disease penetrance due to shortened D4Z4 repeat units on 4q35. The molecular diagnosis of FSHD1 is usually made by Southern blotting, which is complex, time-consuming, and lacks clinical practicality. Therefore, in this study, optical genome mapping (OGM) is employed for the genetic diagnosis of FSHD1. Furthermore, epigenetic heterogeneity is determined from methylation analysis.
Methods
Genomic DNA samples from four members of the same family were subjected to whole-exome sequencing. OGM was used to identify structural variations in D4Z4, while sodium bisulfite sequencing helped identify the methylation levels of CpG sites in a region located distally to the D4Z4 array. A multidisciplinary team collected the clinical data, and comprehensive family analyses aided in the assessment of phenotypes and genotypes.
Results
Whole-exome sequencing did not reveal variants related to clinical phenotypes in the patients. OGM showed that the proband was a compound heterozygote for the 4qA allele with four and eight D4Z4 repeat units, whereas the affected younger brother had only one 4qA allele with four D4Z4 repeat units. Both the proband and her younger brother were found to display asymmetric weakness predominantly involving the facial, shoulder girdle, and upper arm muscles, whereas the younger brother had more severe clinical symptoms. The proband's father, who was found to be normal after a neurological examination, also carried the 4qA allele with eight D4Z4 repeat units. The unaffected mother exhibited 49 D4Z4 repeat units of the 4qA allele and a minor mosaic pattern with four D4Z4 repeat units of the 4qA allele. Consequently, the presence of the 4qA allele in the four D4Z4 repeat units strongly pointed to the occurrence of maternal germline mosaicism. The CpG6 methylation levels were lower in symptomatic patients compared to those in the asymptomatic parents. The older sister had lower clinical scores and ACSS and higher CpG6 methylation levels than that of her younger brother.
Conclusions
In this study, two siblings with FSHD1 with phenotypically normal parents were identified by OGM. Our findings suggest that the 4qA allele of four D4Z4 repeats was inherited through maternal germline mosaicism. The clinical phenotype heterogeneity is influenced by the CpG6 methylation levels. The results of this study greatly aid in the molecular diagnosis of FSHD1 and in also understanding the clinical phenotypic variability underlying the disease.
... Facioscapulohumeral muscular dystrophy (FSHD) is a progressive myopathy that affects women and men of all ages with an estimated worldwide prevalence of ∼1 in 8300-15 000 [1][2][3]. FSHD is caused by aberrant expression of the Double homeobox 4 (DUX4) retrogene in skeletal muscle as a consequence of epigenetic dysregulation of the 4q35 D4Z4 macrosatellite repeat array [4][5][6][7][8]. DUX4 encodes a transcription factor that is expressed at the 4cell stage and plays an important role in the early stages of human embryonic development before becoming epigenetically silenced [9,10]. ...
Facioscapulohumeral muscular dystrophy (FSHD) is a progressive myopathy caused by the aberrant increased expression of the DUX4 retrogene in skeletal muscle cells. The DUX4 gene encodes a transcription factor that functions in zygotic genome activation and then is silenced in most adult somatic tissues. DUX4 expression in FSHD disrupts normal muscle cell function; however, the downstream pathogenic mechanisms are still unclear. Histologically, FSHD affected muscles show a characteristic dystrophic phenotype that is often accompanied by a pronounced immune cell infiltration, but the role of the immune system in FSHD is not understood. Previously, we used ACTA1;FLExDUX4 FSHD-like mouse models varying in severity as discovery tools to identify increased Interleukin 6 and microRNA-206 levels as serum biomarkers for FSHD disease severity. In this study, we use the ACTA1;FLExDUX4 chronic FSHD-like mouse model to provide insight into the immune response to DUX4 expression in skeletal muscles. We demonstrate that these FSHD-like muscles are enriched with the chemoattractant eotaxin and the cytotoxic eosinophil peroxidase, and exhibit muscle eosinophilia. We further identified muscle fibers with positive staining for eosinophil peroxidase in human FSHD muscle. Our data supports that skeletal muscle eosinophilia is a hallmark of FSHD pathology.
... Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscular dystrophy caused by the aberrant expression of the DUX4 gene in skeletal muscle [1]. The reported incidence of FSHD varies across Europe, affecting between 5 and 12 people in 100,000 [2][3][4][5]. ...
Background:
Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disorder characterized by progressive muscle weakness leading to permanent disability. There are no curative treatments, however, there are several upcoming clinical trials testing new therapies in FSHD.
Objective:
This study aimed to explore the disease burden and patient preferences of people with FSHD to ensure that clinical trials can be designed to include outcome measures that are relevant and important to patients.
Methods:
A survey was developed with a steering committee clinicians and physiotherapists with relevant experience in the disease, patient representatives, a registry expert and industry consultants. Themes of the survey included; participant demographics, disease progression and impact on function, factors encouraging or discouraging clinical trial participation, and positive outcomes of a clinical trial.
Results:
1147 participants responded to the online survey, representing 26 countries across Europe and a range of disease severities. The study highlighted the key symptoms causing concern for FSHD patients - muscle weakness and mobility issues - reflecting what participants want targeted for future therapies. The need for clear information and communication throughout clinical trials was emphasised. Factors most encouraging trial participation included access to new investigational therapies, access to trial results and benefits for the FSHD community. Factors most discouraging trial participation included travel related issues and fear of side effects.
Conclusions:
The results from this study identify the patient reported burden of FSHD and should provide researchers and industry with areas of therapeutic research that would be meaningful to patients, as well as supporting the development of patient centric outcome measures in clinical trials.
... Facioscapulohumeral dystrophy (FSHD) is the second most common muscular dystrophy worldwide [1,2] and it is characterized by progressive weakness and atrophy of skeletal muscles [3]. ...
Background:
FSHD is a highly prevalent inherited myopathy with a still poorly understood pathology.
Objective:
To investigate whether proinflammatory cytokines are associated with FSHD and which specific innate immune cells are involved in its pathology.
Methods:
First, we measured circulating cytokines in serum samples: IL-6 (FSHD, n = 150; HC, n = 98); TNF (FSHD, n = 150; HC, n = 59); IL-1α (FSHD, n = 150; HC, n = 66); IL-1β (FSHD, n = 150; HC, n = 98); MCP-1 (FSHD, n = 14; HC, n = 14); VEGF-A (FSHD, n = 14; HC, n = 14). Second, we tested trained immunity in monocytes (FSHD, n = 15; HC, n = 15) and NK cells (FSHD, n = 11; HC, n = 11). Next, we explored the cytokine production capacity of NK cells in response to different stimuli (FSHD, n = 39; HC, n = 22). Lastly, we evaluated the cytokine production of ex vivo stimulated MRI guided inflamed (TIRM+) and paired MRI guided non inflamed (TIRM-) muscle biopsies of 21 patients and of 8 HC muscle biopsies.
Results:
We included a total of 190 FSHD patients (N = 190, 48±14 years, 49% men) and of 135 HC (N = 135, 44±15 years, 47% men). We found that FSHD patients had higher concentrations of IL-6 and TNF measured (a) in the circulation, (b) after ex-vivo stimulation of NK cells, and (c) in muscle specimens. Besides, IL-6 circulating concentrations, as well as its production by NK cells and IL-6 content of FSHD muscle specimens, showed a mild correlation with disease duration, disease severity, and muscle weakness.
Conclusion:
These results show that IL-6 and TNF may contribute to FSHD pathology and suggest novel therapeutic targets. Additionally, the activation of NK cells in FSHD may be a novel pathway contributing to FSHD pathology.
... Facioscapulohumeral dystrophy (FSHD) is a common autosomal dominant muscular dystrophy with international prevalence estimates ranging from 0.8 to 12/100 000 1,2 . There are two genetic subtypes (FSHD1 and FSHD2), which have a common pathophysiological mechanism of ectopic expression of the DUX4 gene in skeletal muscle stemming from hypomethylation of the D4Z4 array [3][4][5] . ...
Background and Objectives: Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease causing changes in muscle structure that can negatively affect upper-limb function. Echogenicity, measured using quantified muscle ultrasound, is a potential biomarker that could be used for informing decision making. Histogram-matching allows for image normalisation, which could enable comparison of echogenicity between different machine capture settings which is a current limitation. This study aimed to investigate if ultrasonography and histogram-matching can measure trapezius muscle echogenicity and morphology for differentiating between people with and without FSHD, and different levels of arm function.
Methods: Single measurement timepoint case control study of adults with FSHD and age- and sex- matched controls. Main outcomes were trapezius echogenicity and muscle thickness measured using 2D-ultrasound, and maximum thoracohumeral elevation angle, measured using 3D-movement analysis. A sensitivity analysis evaluating the effect of histogram-matching and different reference images was conducted. Between group differences for echogenicity were evaluated using an unpaired student t-test. Echogenicity, muscle thickness and range of movement were plotted to evaluate the explained variance between variables.
Results: Data was collected for 14 participants (10M:4F), seven with FSHD and seven controls with a mean (SD) age of 41.6 (15.7). Normalisation was necessary and echogenicity values for the FSHD group were higher than the controls (118.2 (34.0) vs 42.3 (14.0) respectively, with statistically significant differences (p=0.002). An overall variance of 6.2 (LLOA -2.9 to ULOA 15.4) was identified between reference images. Echogenicity accounted for the largest explained variance in muscle thickness (R2=0.81) and range of movement (R2=0.74), whilst muscle thickness and range of movement was the lowest (R2=0.61).
Discussion: People with FSHD demonstrated higher echogenicity, smaller muscle thicknesses and less range of movement. Histogram-matching for comparison of echogenicity values is necessary and can provide quantifiable differences. Different reference images affect echogenicity values but the variability is less than between group differences. Further work is needed to evaluate the longitudinal variability associated with this method on a larger sample of people with varying levels of arm function. Ultrasound scanning and post-histogram matching may be used to quantify and compare differences in muscle structure and function people with and without FSHD.
... 3-6 Importantly, variants in FSHD2 genes can also act as disease modifiers in the presence of D4Z4 Reduced Allele (DRA) < 10 RU. 4,6 The estimated prevalence of FSHD ranges from 1:8000 to 1:20.000. 1,7 Although the mode of inheritance is autosomal dominant, 10%-30% of cases are sporadic. 8,9 Several Mendelian disorders show approximately 7%-10% of sporadic cases explained by de novo genetic alterations, while other conditions (Duchenne Muscular Dystrophy) reports higher rates (33%). ...
Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant disease, although 10%–30% of cases are sporadic. However, this percentage may include truly de novo patients (carrying a reduced D4Z4 allele that is not present in either of the parents) and patients with apparently sporadic disease resulting from mosaicism, non‐penetrance, or complex genetic situations in either patients or parents. In this study, we characterized the D4Z4 Reduced Alleles (DRA) and evaluated the frequency of truly de novo cases in FSHD1 in a cohort of DNA samples received consecutively for FSHD‐diagnostic from 100 Italian families. The D4Z4 testing revealed that 60 families reported a DRA compatible with FSHD1 (1–10 RU). The DRA co‐segregated with the disease in most cases. Five families with truly de novo cases were identified, suggesting that this condition may be slightly lower (8%) than previously reported. In addition, D4Z4 characterization in the investigated families showed 4% of mosaic cases and 2% with translocations. This study further highlighted the importance of performing family studies for clarifying apparently sporadic FSHD cases, with significant implications for genetic counseling, diagnosis, clinical management, and procreative choices for patients and families.
... With an estimated prevalence of nearly 1 in 8000 individuals [1] Facioscapulohumeral Muscular Dystrophy (FSHD) is one of the most common muscular dystrophies. Pathology typically begins with weakness in the facial, scapular, and humeral muscles, but progresses to the trunk and lower extremities, resulting in profound disability [2]. ...
Facioscapulohumeral muscular dystrophy (FSHD) is among the most common of the muscular dystrophies, affecting nearly 1 in 8000 individuals, and is a cause of profound disability. Genetically, FSHD is linked to the contraction and/or epigenetic de-repression of the D4Z4 repeat array on chromosome 4, thereby allowing expression of the DUX4 gene in skeletal muscle. If the DUX4 transcript incorporates a stabilizing polyadenylation site the myotoxic DUX4 protein will be synthesized, resulting in muscle wasting. The mechanism of toxicity remains unclear, as many DUX4-induced cytopathologies have been described, however cell death does primarily occur through caspase 3/7-dependent apoptosis. To date, most FSHD therapeutic development has focused on molecular methods targeting DUX4 expression or the DUX4 transcript, while therapies targeting processes downstream of DUX4 activity have received less attention. Several studies have demonstrated that inhibition of multiple signal transduction pathways can ameliorate DUX4-induced toxicity, and thus compounds targeting these pathways have the potential to be developed into FSHD therapeutics. To this end, we have screened a group of small molecules curated based on their reported activity in relevant pathways and/or structural relationships with known toxicity-modulating molecules. We have identified a panel of five compounds that function downstream of DUX4 activity to inhibit DUX4-induced toxicity. Unexpectedly, this effect was mediated through an mTor-independent mechanism that preserved expression of ULK1 and correlated with an increase in a marker of active cellular autophagy. This identifies these flavones as compounds of interest for therapeutic development, and potentially identifies the autophagy pathway as a target for therapeutics.
... Facioscapulohumeral muscular dystrophy (FSHD) is the second most common muscular dystrophy in adults, with a prevalence of nearly 1:8000 [1]. It causes muscle weakness initially affecting the muscles in the face and around the scapula and then progressively affecting the arm and legs. ...
Facioscapulohumeral muscular dystrophy (FSHD) is the second most common muscular dystrophy in adults, and it is associated with local D4Z4 chromatin relaxation, mostly via the contraction of the D4Z4 macrosatellite repeat array on chromosome 4q35. In this study, we aimed to investigate the use of Optical Genome Mapping (OGM) as a diagnostic tool for testing FSHD cases from the UK and India and to compare OGM performance with that of traditional techniques such as linear gel (LGE) and Pulsed-field gel electrophoresis (PFGE) Southern blotting (SB). A total of 6 confirmed and 19 suspected FSHD samples were processed with LGE and PFGE, respectively. The same samples were run using a Saphyr Genome-Imaging Instrument (1-color), and the data were analysed using custom EnFocus FSHD analysis. OGM was able to confirm the diagnosis of FSHD1 in all FSHD1 cases positive for SB (n = 17), and D4Z4 sizing highly correlated with PFGE-SB (p < 0.001). OGM correctly identified cases with mosaicism for the repeat array contraction (n = 2) and with a duplication of the D4Z4 repeat array. OGM is a promising new technology able to unravel structural variants in the genome and seems to be a valid tool for diagnosing FSHD1.
... Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy with a prevalence of ∼12/100 000 [1] that typically presents during the second or third decade of life. FSHD is characterized by muscle weakness and a progressive loss of muscle mass that typically begins in the facial muscles before progressing to the shoulder girdle and lower limb [2][3][4]. ...
Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy. FSHD is highly heterogeneous, with patients following a variety of clinical trajectories, complicating clinical trials. Skeletal muscle in FSHD undergoes fibrosis and fatty replacement that can be accelerated by inflammation, adding to heterogeneity. Well controlled molecular studies are thus essential to both categorise FSHD patients into distinct subtypes and understand pathomechanisms. Here, we further analysed RNA-sequencing data from 24 FSHD patients, each of whom donated a biopsy from both a non-inflamed (TIRM−) and inflamed (TIRM+) muscle, and 15 FSHD patients who donated peripheral blood mononucleated cells (PBMCs), alongside non-affected control individuals. Differential gene expression analysis identified suppression of mitochondrial biogenesis and up-regulation of fibroadipogenic progenitor (FAP) gene expression in FSHD muscle, which was particularly marked on inflamed samples. PBMCs demonstrated suppression of antigen presentation in FSHD. Gene expression deconvolution revealed FAP expansion as a consistent feature of FSHD muscle, via meta-analysis of 7 independent transcriptomic datasets. Clustering of muscle biopsies separated patients in an unbiased manner into clinically mild and severe subtypes, independently of known disease modifiers (age, sex, D4Z4 repeat length). Lastly, the first genome-wide analysis of alternative splicing in FSHD muscle revealed perturbation of autophagy, BMP2 and HMGB1 signalling. Overall, our findings reveal molecular subtypes of FSHD with clinical relevance and identify novel pathomechanisms for this highly heterogeneous condition.
... Facioscapulohumeral muscular dystrophy (FSHD) is a muscular dystrophy characterized by the progressive weakening and atrophy of muscle groups primarily in, but not restricted to, the face (facio), around the shoulder blades (scapula), and in the upper arms (humeral) [1]. This incurable, genetic disease is one of the most prevalent muscular dystrophies, afflicting an estimated 12/100,000 people [2]. Though numerous strategies have been developed to manage symptoms, there are currently no approved treatments for FSHD. ...
Facioscapulohumeral dystrophy (FSHD) is a muscle disease caused by inappropriate expression of the double homeobox 4 (DUX4) gene in skeletal muscle, and its downstream activation of pro-apoptotic transcriptional programs. Inhibitors of DUX4 expression have the potential to treat FSHD. Apabetalone is a clinical-stage bromodomain and extra-terminal (BET) inhibitor, selective for the second bromodomain on BET proteins. Using primary human skeletal muscle cells from FSHD type 1 patients, we evaluated apabetalone for its ability to counter DUX4′s deleterious effects and compared it with the pan-BET inhibitor JQ1, and the p38 MAPK inhibitor—and DUX4 transcriptional repressor—losmapimod. We applied RNA-sequencing and bioinformatic analysis to detect treatment-associated impacts on the transcriptome of these cells. Apabetalone inhibited the expression of DUX4 downstream markers, reversing hallmarks of FSHD gene expression in differentiated muscle cells. JQ1, but not apabetalone, was found to induce apoptosis. While both BET inhibitors modestly impacted differentiation marker expression, they did not affect myotube fusion. Losmapimod also reduced expression of DUX4 target genes but differed in its impact on FSHD-associated pathways. These findings demonstrate that apabetalone inhibits DUX4 target gene expression and reverses transcriptional programs that contribute to FSHD pathology, making this drug a promising candidate therapeutic for FSHD.
... FSHD (OMIM #158900, #158901, #619477, #619478) is a skeletal muscle disorder with an estimated prevalence of 1:8000-20.000 (Mostacciuolo et al., 2009;Deenen et al., 2014). A progressive and often asymmetric weakness of facial, shoulder and upper arm muscles are typical features of disease, although abdominal, hip girdle and lower leg muscles are also frequently involved (Preston et al., 2020). ...
Introduction: Despite the progress made in the study of Facioscapulohumeral Dystrophy (FSHD), the wide heterogeneity of disease complicates its diagnosis and the genotype-phenotype correlation among patients and within families. In this context, the present work employed Whole Exome Sequencing (WES) to investigate known and unknown genetic contributors that may be involved in FSHD and may represent potential disease modifiers, even in presence of a D4Z4 Reduced Allele (DRA).
Methods: A cohort of 126 patients with clinical signs of FSHD were included in the study, which were characterized by D4Z4 sizing, methylation analysis and WES. Specific protocols were employed for D4Z4 sizing and methylation analysis, whereas the Illumina® Next-Seq 550 system was utilized for WES. The study included both patients with a DRA compatible with FSHD diagnosis and patients with longer D4Z4 alleles. In case of patients harboring relevant variants from WES, the molecular analysis was extended to the family members.
Results: The WES data analysis highlighted 20 relevant variants, among which 14 were located in known genetic modifiers (SMCHD1, DNMT3B and LRIF1) and 6 in candidate genes (CTCF, DNMT1, DNMT3A, EZH2 and SUV39H1). Most of them were found together with a permissive short (4–7 RU) or borderline/long DRA (8–20 RU), supporting the possibility that different genes can contribute to disease heterogeneity in presence of a FSHD permissive background. The segregation and methylation analysis among family members, together with clinical findings, provided a more comprehensive picture of patients.
Discussion: Our results support FSHD pathomechanism being complex with a multigenic contribution by several known (SMCHD1, DNMT3B, LRIF1) and possibly other candidate genes (CTCF, DNMT1, DNMT3A, EZH2, SUV39H1) to disease penetrance and expressivity. Our results further emphasize the importance of extending the analysis of molecular findings within the proband’s family, with the purpose of providing a broader framework for understanding single cases and allowing finer genotype-phenotype correlations in FSHD-affected families.
... Symptoms of FSHD include weakness in the muscles of the face, shoulders, and upper arms, and can include limb and trunk muscles as the disease progresses [1][2][3]. It has a prevalence of 1 in 8333-20,000 people in the world [1,[4][5][6][7]. Current therapies for FSHD only address its symptoms, such as shoulder fixation orthotics and physical therapy [2]. ...
Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the double homeobox 4 (DUX4) gene. The FLExDUX4 mouse model carries an inverted human DUX4 transgene which has leaky DUX4 transgene expression at a very low level. No overt muscle pathology was reported before 16 weeks. The purpose of this study is to track and characterize the FLExDUX4 phenotypes for a longer period, up to one year old. In addition, transcriptomic changes in the muscles of 2-month-old mice were investigated using RNA-seq. The results showed that male FLExDUX4 mice developed more severe phenotypes and at a younger age in comparison to the female mice. These include lower body and muscle weight, and muscle weakness measured by grip strength measurements. Muscle pathological changes were observed at older ages, including fibrosis, decreased size of type IIa and IIx myofibers, and the development of aggregates containing TDP-43 in type IIb myofibers. Muscle transcriptomic data identified early molecular changes in biological pathways regulating circadian rhythm and adipogenesis. The study suggests a slow progressive change in molecular and muscle phenotypes in response to the low level of DUX4 expression in the FLExDUX4 mice.
BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is often characterized by weakness of the scapular stabilizing muscles. FSHD is a genetic disorder in which skeletal muscles of the face (facio), the shoulder blades (scapulo), arms (humeral), and other muscles progressively weaken over time. ⁵
CASE PRESENTATION: A 13-year-old tennis player developed progressive right shoulder pain and limited range of motion. He did not have a precedent injury and his symptoms were exacerbated by tennis. His exam was notable for medial scapular winging. An electromyogram was read as a long thoracic nerve palsy, presumed to be a tennis-related traction injury. He was treated with aggressive therapy, taping, and bracing to improve scapulothoracic and glenohumeral biomechanics for optimal upper extremity strength and range of motion.
OUTCOME AND FOLLOW-UP: His pain and motion improved, but he was unable to return to his prior level of tennis competition. Eighteen months after the initial diagnosis, the patient presented with similar symptoms and findings on the contralateral side, raising concern for a neuromuscular etiology.
DISCUSSION: A repeat electromyogram and genetic tests were consistent with FSHD, rather than a sport-related peripheral nerve injury. JOSPT Cases 2023;3(2):96–99. Epub: 13 March 2023. doi:10.2519/josptcases.2023.10887
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common autosomal dominant muscle disorders, yet no cure or amelioration exists. The clinical presentation is diverse, making it difficult to identify the actual driving pathomechanism among many downstream events. To unravel this complexity, we performed a meta-analysis of 13 original omics datasets (in total 171 FSHD and 129 control samples). Our approach confirmed previous findings about the disease pathology and specified them further. We confirmed increased expression of former proposed DUX4 biomarkers, and furthermore impairment of the respiratory chain. Notably, the meta-analysis provides insights about so far not reported pathways, including misregulation of neuromuscular junction protein encoding genes, downregulation of the spliceosome, and extensive alterations of nuclear envelope protein expression. Finally, we developed a publicly available shiny app to provide a platform for researchers who want to search our analysis for genes of interest in the future.
Neuromuscular diseases encompass a heterogeneous array of disorders characterized by varying onset ages, clinical presentations, severity, and progression. While these conditions can stem from acquired or inherited causes, this review specifically focuses on disorders arising from genetic abnormalities, excluding metabolic conditions. The pathogenic defect may primarily affect the anterior horn cells, the axonal or myelin component of peripheral nerves, the neuromuscular junction, or skeletal and/or cardiac muscles. While inherited neuromuscular disorders have been historically deemed not treatable, the advent of gene-based and molecular therapies is reshaping the treatment landscape for this group of condition. With the caveat that many products still fail to translate the positive results obtained in pre-clinical models to humans, both the technological development (e.g., implementation of tissue-specific vectors) as well as advances on the knowledge of pathogenetic mechanisms form a collective foundation for potentially curative approaches to these debilitating conditions. This review delineates the current panorama of therapies targeting the most prevalent forms of inherited neuromuscular diseases, emphasizing approved treatments and those already undergoing human testing, offering insights into the state-of-the-art interventions.
Purpose:
To develop a multidisciplinary outpatient rehabilitation intervention for people with neuromuscular diseases (NMD) based on the capability approach: capability care for persons with NMD.
Materials and methods:
The development process is described using a framework of actions for intervention development. It has been an iterative process consisting of a design phase based on theoretical insights and project group discussions, and a refine phase involving input from relevant stakeholders.
Results:
Multidisciplinary efforts have resulted in the development of capability care for rehabilitation of persons with NMD. It can focus both on facilitating and achieving functionings (beings and doings), as well as looking for alternative functionings that fulfil the same underlying value, thereby contributing to the persons' well-being. To facilitate a conversation on broader aspects that impact on well-being, persons with NMD receive a preparation letter and healthcare professionals are provided with guiding questions and practical tools to use.
Conclusions:
We have shown that it is possible to develop a healthcare intervention based on the capability approach. We hope that rehabilitation professionals will be encouraged to use capability care and that other medical professionals will be inspired to develop capability care in their respective fields.
Registration:
Registered at trialregister.nl NL8946.
Purpose of review
This review aims to provide a summary of the pathophysiology, clinical presentation and management options for facioscapulohumeral dystrophy (FSHD). We discuss current management options and delve into updates about developments in targeted therapy.
Recent findings
New breakthroughs in FSHD research have led to a further understanding of aberrant DUX4 protein expression in the underlying pathophysiology of FSHD. This has paved the way for the development of targeted therapies aimed at targeting DUX4 expression or its downstream effects. Therapeutic strategies for FSHD primarily target DUX4 through three main avenues: small molecules, antisense oligonucleotide therapeutics and CRISPR-based approaches. This review discusses these strategies further. Presently, all prospective targeted therapies are in the pre-clinical phase, except for losmapimod, which is currently undergoing a phase 3 clinical trial.
Summary
Given the absence of approved disease-modifying treatments for FSHD, the primary approach for management currently involves multidisciplinary supportive measures which are limited. Recent developments in the form of targeted therapies and strategies for the definitive treatment of FSHD indicate a promising era.
Cell therapies derived from induced pluripotent stem cells (iPSCs) offer a promising avenue in the field of regenerative medicine due to iPSCs’ expandability, immune compatibility, and pluripotent potential. An increasing number of preclinical and clinical trials have been carried out, exploring the application of iPSC-based therapies for challenging diseases, such as muscular dystrophies. The unique syncytial nature of skeletal muscle allows stem/progenitor cells to integrate, forming new myonuclei and restoring the expression of genes affected by myopathies. This characteristic makes genome-editing techniques especially attractive in these therapies. With genetic modification and iPSC lineage specification methodologies, immune-compatible healthy iPSC-derived muscle cells can be manufactured to reverse the progression of muscle diseases or facilitate tissue regeneration. Despite this exciting advancement, much of the development of iPSC-based therapies for muscle diseases and tissue regeneration is limited to academic settings, with no successful clinical translation reported. The unknown differentiation process in vivo, potential tumorgenicity, and epigenetic abnormality of transplanted cells are preventing their clinical application. In this review, we give an overview on preclinical development of iPSC-derived myogenic cell transplantation therapies including processes related to iPSC-derived myogenic cells such as differentiation, scaling-up, delivery, and cGMP compliance. And we discuss the potential challenges of each step of clinical translation. Additionally, preclinical model systems for testing myogenic cells intended for clinical applications are described.
Purpose:
The Facioscapulohumeral Muscular Dystrophy Health Index (FSHD-HI) is a patient-reported outcome measure developed for patients with FSHD. This study aimed to translate the FSHD-HI into Japanese (FSHD-HI-J), evaluate cultural adaptation, and examine its psychometric properties.
Materials and methods:
We created two forward translations, integrated them into a single Japanese version, and evaluated the back-translated version of the FSHD-HI. After finalizing the translation and cultural adaptation, we conducted a survey of 66 patients with FSHD to examine the reliability and validity of the FSHD-HI-J. For psychometric evaluations, we used Cronbach's alpha to assess internal consistency, the intraclass correlation coefficient (ICC) for test-retest reliability, and assessed validity based on the associations between FSHD-HI-J, clinical variables, and quality of life measures.
Results:
The FSHD-HI-J was found to be clinically relevant, indicating high internal consistency and test-retest reliability (ICC = 0.92 [95% confidence interval: 0.86-0.95] for the total score), as well as significant associations with clinical variables (D4Z4 repeats and functional impairment) and other quality of life measures (|rho| = 0.25-0.73).
Conclusions:
The FSHD-HI-J is a valid and reliable patient-reported outcome measure for Japanese patients with FSHD. This validated, disease-specific patient-reported outcome is essential for future clinical practice and clinical trials.
Fasiyo-skapulo-humeral musküler distrofi yüz, skapular, üst kol, alt bacak ve kalça kuşağı kaslarını içeren, genellikle asimetrik tutulumla birlikte yavaş ilerleyen kas zayıflığı ile karakterize bir genetik bozukluktur. Semptomatik olanlarda her zaman olmasa da sıklıkla en fazla 5 kata kadar kreatinin kinaz seviyesi yükselir. Elektromiyografi miyopatik özellikler gösterir. Kas biyopsisinde nonspesifik miyopatik değişiklikler izlenir. Tanıda deltoidlerin göreceli olarak korunduğu yüz, omuz kuşağı ve üst kol zayıflığı ile başvuran hastalarda şüphelenilmelidir. Genetik analiz tanıda temel yöntemdir. Özellikle tedaviye yanıtsız dermatomiyozit hastalarında tanı tekrar gözden geçirilmeli, özellikle asimetrik ve distal kas güçsüzlüğünün olduğu hastalarda dermatomiyozit tanısından uzaklaşılmalı, yüz ve skapular kas güçsüzlüğünün eşlik ettiği hastalarda akla Fasiyo-skapulo-humeral musküler distrofi gelmelidir.
Scapulothoracic arthrodesis (STA) surgery is performed to stabilize the scapula in patients with facioscapulohumeral dystrophy (FSHD). Postoperative pain could be a major problem even while using erector spinae plane block (ESPB). We performed a preoperative ESPB with an intraoperative ESPB catheter, but rescue analgesia was needed for pain in the periscapular area in the postoperative period. The patient’s pain score was reduced by applying an ultrasound-guided dorsal scapular nerve (DSN) and long thoracic nerve (LTN) block. Selective DSN and LTN blocks can be effective in enhancing postoperative analgesia in STA surgery.
Muscular dystrophies are a large group of genetic muscle diseases, and their diagnosis has become easier, faster, more reliable, and more economical since around 2015. The advent of next generation sequencing has decreased the time and cost of genetic testing by several orders of magnitude, allowing genetic testing early in patient evaluations, often at the first clinic visit. Exome and genome sequencing further increase diagnostic yields; however, this benefit is counterbalanced by data glut, the generation of many variants of uncertain significance.
Genetic counseling is integral to the genetic testing process, as counselors ensure patients are informed and prepared for the possible results. Genetic counselors interactively educate patients and families on basic genetics; possible results; potential emotional, social, and health management impacts for the patient; implications for family members; and even options around future reproductive planning.
With the advent of gene-based therapies, genetic counseling and testing are now of paramount importance in the practice of medicine. With genome sequencing at birth already available as newborn screening in some healthcare systems, personalized genetics will soon serve as the template for health throughout the lifespan.
Facioscapulohumeral muscular dystrophy is the most prevalent dominantly-inherited muscular dystrophy after myotonic dystrophy. The most distinctive clinical features are the characteristic facial weakness and scapular winging. Whereas the pattern of muscle involvement is similar in many patients, the age at onset, rate of progression and overall disease severity is very variable. Symptomatic extra-muscular manifestations such as retinal exudative retinopathy or hearing loss are rare and limited to severely affected, infantile onset disease. The genetic cause of this dystrophy in the majority of cases is the epigenetic activation of the DUX4 gene, a gene normally only expressed in early development but not in somatic cells. The ectopic expression of DUX4, a transcription factor, in myofibers results in the activation of a number of deleterious mechanisms resulting in muscle weakness. No approved targeted treatment exists for facioscapulohumeral muscular dystrophy. However, the underlying disease mechanism is amenable to treatment using targeted antisense oligonucleotides, an approach being actively pursued by several pharmaceutical companies.
The molecular diagnosis of type 1 FacioScapuloHumeral Dystrophy (FSHD1) relies on the detection of a shortened D4Z4 array at the 4q35 locus while until recently, the diagnosis of FSHD2 relied on the absence of a shortened D4Z4 allele in clinically affected patients. The vast majority of FSHD2 patients carry a heterozygous variant in the SMCHD1 gene. In addition, a decreased in D4Z4 DNA methylation is consistently associated with FSHD1 and FSHD2. In molecular genetic diagnostics, predicting the pathogenicity of SMCHD 1 variants remains challenging, as many are classified as variants of unknown significance or likely pathogenic. To refine the diagnosis of FSHD2, define 4q-associated molecular features and validate the pathogenicity of SMCHD1 variants, we explored a cohort of 54 FSHD2 patients carrying a variant in SMCHD1 or hemizygosity of the 18p32 locus encompassing the gene. Genetic and epigenetic analyses together with a clinical description of patients were combined to confirm the pathogenicity of new SMCHD1 variants and previously reported ones initially classified as likely pathogenic. We defined a threshold of 40% of methylation at the D4Z4 DR1 site as associated with SMCHD1 pathogenic variants. We also showed that the number of D4Z4 units on the shortest 4qA allele ranges from 11 up to 35 units in patients clinically affected with FSHD2. Using prediction tools, our study further highlighted the difficulty in interpretating the impact of pathogenic variants on the severity of the disease. Our study further emphasizes the complex relationship between D4Z4 methylation, SMCHD1 variants, and disease penetrance in FSHD.
The study is to explore the feasibility and value of SNP‐based noninvasive prenatal diagnosis (NIPD) for facioscapulohumeral muscular dystrophy type 1 (FSHD1) in early pregnancy weeks. We prospectively collected seven FSHD1 families, with an average gestational age of 8 ⁺⁶ . Among these seven couples, there were three affected FSHD1 mothers and four affected fathers. A multiplex‐PCR panel comprising 402 amplicons was designed to selective enrich for highly heterozygous SNPs upstream of the DUX4 gene. Risk haplotype was constructed based on familial linkage analysis. Fetal genotypes were accurately inferred through relative haplotype dosage analysis using Bayes Factor. All tests were successfully completed in a single attempt, and no recombination events were detected. NIPD results were provided within a week, which is 4 weeks earlier than karyomapping and 7 weeks earlier than Bionano single‐molecule optical mapping (BOM). Ultimately, five FSHD1 fetuses and two normal fetuses were successfully identified, with a 100% concordance rate with karyomapping and BOM. Therefore, SNP‐based NIPD for FSHD1 was demonstrated to be feasible and accurate in early weeks of gestation, although the risk of recombination events cannot be completely eliminated. In the future, testing of more cases is still necessary to fully determine the clinical utility.
Background
Facioscapulohumeral dystrophy (FSHD) almost always affects the muscles of the face. Quantiative evalution of face is essential for the identification of severity in the facial region. The aim of this study is to evaluate facial involvement of FSHD patients with three-dimensional(3D) scanning to answer these questions: (Q1) Are the facial 3D evaluations of FSHD patients different compared to healthy individuals? (Q2) Do 3D evaluations correlate with genetic mutation and clinical severity of the disease?
Methods
The 3D facial scanning was performed in three axes coordinate system. Outcome variables were intensity and asymmetry estimations of those facial expressions; eyebrow elevation, maximal closing of the eyes, maximal showing of the teeth, whistling, and happy. Genetic diagnosis was performed with southern blot analysis to reveal D4Z4 repeat contraction on 4q35 (D4Z4-RU). Clinical severity score (CSS) was determined via neurological examination. For Q1, intensity and asymmetry related 3D variables were compared between the control and FSHD groups. For Q2, the correlation analysis was performed between intensity related 3D variables and genetic mutation and clinical severity. The level of significance had been set to p ≤ 0. 05.
Results
Twenty-one healthy controls (57.1% female) and 12 FSHD patients (58.3% female) were included. Significantly different (p < 0.002 and < 0.001) facial expressions were; the intensity of whistling, maximal closing of the eyes and happy expressions. There was no significant difference in asymmetry related measurements. No correlation between facial variables with D4Z4-RU and CSS was detected.
Conclusions
The 3D method can be used as a quantitative evaluation method in diseases manifested by impaired mimic muscle functions such as FSHD.
Introduction/Aims
In preparation for clinical trials, it is important to better understand how disease burden changes over time in facioscapulohumeral muscular dystrophy (FSHD) and to assess the capability of select metrics to detect these changes. This study aims to evaluate FSHD disease progression over 1 year and to examine the sensitivity of several outcome measures in detecting changes during this interval.
Methods
We conducted a 12‐month prospective observational study of 41 participants with FSHD. Participants were evaluated at baseline, 6 months, and 12 months with serial strength testing (manual muscle testing or MMT and maximum voluntary isometric contraction testing or MVICT), functional testing (FSHD‐Composite Outcome Measure or FSHD‐COM, FSHD Clinical Severity Score or CSS, and FSHD Evaluation Score or FES), sleep and fatigue assessments, lean body mass measurements, respiratory testing, and the FSHD‐Health Index patient‐reported outcome. Changes in these outcome measures were assessed over the 12‐month period. Associations between changes in outcome measures and both age and sex were also examined.
Results
In a 12‐month period, FSHD participant function remained largely stable with a mild worsening of strength, measured by MMT and standardized MVICT scores, and a mild loss in lean body mass.
Discussion
The abilities and disease burden of adults with FSHD are largely static over a 12‐month period with participants demonstrating a mild average reduction in some measures of strength. Selection of patients, outcome measures, and trial duration should be carefully considered during the design and implementation of future clinical studies involving FSHD patients.
Background and Objectives
Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common muscular dystrophy in the general population and is characterized by progressive and often asymmetric muscle weakness of the face, upper extremities, arms, lower leg, and hip girdle. In FSHD type 1, contraction of the number of D4Z4 repeats to 1–10 on the chromosome 4–permissive allele (4qA) results in abnormal epigenetic derepression of the DUX4 gene in skeletal muscle. In FSHD type 2, epigenetic derepression of the DUX4 gene on the permissive allele (4qA) with normal-sized D4Z4 repeats (mostly 8–20) is caused by heterozygous pathogenic variants in chromatin modifier genes such as SMCHD1 , DNMT3B , or LRIF1 . We present validation of the optical genome mapping (OGM) platform for accurate mapping of the D4Z4 repeat size, followed by diagnostic testing of 547 cases with a suspected clinical diagnosis of FSHD and next-generation sequencing (NGS) of the SMCHD1 gene to identify cases with FSHD2.
Methods
OGM with Bionano Genomics Saphyr and EnFocus FSHD analysis software was used to identify FSHD haplotypes and D4Z4 repeat number and compared with the gold standard of Southern blot–based diagnosis. A custom Agilent SureSelect enrichment kit was used to enrich SMCHD1 , followed by NGS on an Illumina system with 100-bp paired-end reads. Copy number variants were assessed using NxClinical software.
Results
We performed OGM for the diagnosis of FSHD in 547 patients suspected of FSHD between December 2019 and December 2022, including 301 male (55%) and 246 female patients (45%). Overall, 308 of the referred patients were positive for D4Z4 contraction on a permissive haplotype, resulting in a diagnosis of FSHD1. A total of 252 of 547 patients were referred for concurrent testing for FSHD1 and FSHD2. This resulted in the identification of FSHD2 in 9/252 (3.6%) patients. In our FSHD2 cohort, the 4qA allele size ranged from 8 to 18 repeats. Among FSHD1-positive cases, 2 patients had biallelic contraction and 4 patients had homozygous contraction and showed early onset of clinical features. Nine of the 308 patients (3%) positive for 4qA contraction had mosaic 4q alleles with contraction on at least one 4qA allele. The overall diagnostic yield in our cohort was 58%.
Discussion
A combination of OGM to identify the FSHD haplotype and D4Z4 repeat number and NGS to identify sequence and copy number variants in the SMCHD1 gene is a practical and cost-effective option with increased precision for accurate diagnosis of FSHD types 1 and 2.
Fascioscapulohumeral muscular dystrophy (FSHD) is caused by a unique genetic mechanism that relies on contraction and hypomethylation of the D4Z4 macrosatellite array on the Chromosome 4q telomere allowing ectopic expression of the DUX4 gene in skeletal muscle. Genetic analysis is difficult because of the large size and repetitive nature of the array, a nearly identical array on the 10q telomere, and the presence of divergent D4Z4 arrays scattered throughout the genome. Here, we combine nanopore long-read sequencing with Cas9-targeted enrichment of 4q and 10q D4Z4 arrays for comprehensive genetic analysis including determination of the length of the 4q and 10q D4Z4 arrays with base-pair resolution. In the same assay, we differentiate 4q from 10q telomeric sequences, determine A/B haplotype, identify paralogous D4Z4 sequences elsewhere in the genome, and estimate methylation for all CpGs in the array. Asymmetric, length-dependent methylation gradients were observed in the 4q and 10q D4Z4 arrays that reach a hypermethylation point at approximately 10 D4Z4 repeat units, consistent with the known threshold of pathogenic D4Z4 contractions. High resolution analysis of individual D4Z4 repeat methylation revealed areas of low methylation near the CTCF/insulator region and areas of high methylation immediately preceding the DUX4 transcriptional start site. Within the DUX4 exons, we observed a waxing/waning methylation pattern with a 180-nucleotide periodicity, consistent with phased nucleosomes. Targeted nanopore sequencing complements recently developed molecular combing and optical mapping approaches to genetic analysis for FSHD by adding precision of the length measurement, base-pair resolution sequencing, and quantitative methylation analysis.
Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable skeletal myopathy. Clinical trials for FSHD are hindered by heterogeneous biomarkers poorly associated with clinical severity, requiring invasive muscle biopsy. Macroscopically, FSHD presents with slow fatty replacement of muscle, rapidly accelerated by inflammation. Mis-expression of the transcription factor DUX4 is currently accepted to underlie pathogenesis, and mechanisms including PAX7 target gene repression have been proposed. Here, we performed RNA-sequencing on MRI-guided inflamed and isogenic non-inflamed muscle biopsies from the same clinically characterized FSHD patients (n = 24), alongside isogenic peripheral blood mononucleated cells from a subset of patients (n = 13) and unaffected controls (n = 11). Multivariate models were employed to evaluate the clinical associations of five published FSHD transcriptomic biomarkers. We demonstrated that PAX7 target gene repression can discriminate control, inflamed and non-inflamed FSHD muscle independently of age and sex (P < 0.013), while the discriminatory power of DUX4 target genes was limited to distinguishing FSHD muscle from control. Importantly, the level of PAX7 target gene repression in non-inflamed muscle associated with clinical assessments of FSHD severity (P = 0.04). DUX4 target gene biomarkers in FSHD muscle showed associations with lower limb fat fraction and D4Z4 array length but not clinical assessment. Lastly, PAX7 target gene repression in FSHD muscle correlated with the level in isogenic peripheral blood mononucleated cells (P = 0.002). A refined PAX7 target gene biomarker comprising 143/601 PAX7 target genes computed in peripheral blood (the FSHD muscle–blood biomarker) associated with clinical severity in FSHD patients (P < 0.036). Our new circulating biomarker validates as a classifier of clinical severity in an independent data set of 54 FSHD patient and 29 matched control blood samples, with improved power in older patients (P = 0.03). In summary, we present the minimally invasive FSHD muscle–blood biomarker of FSHD clinical severity valid in patient muscle and blood, of potential use in routine disease monitoring and clinical trials.
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular disorders and has no cure. Due to an unknown molecular mechanism, FSHD displays overlapping manifestations with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). FSHD is caused by aberrant gain of expression of the transcription factor double homeobox 4 (DUX4), which triggers a pro-apoptotic transcriptional program resulting in inhibition of myogenic differentiation and muscle wasting. Regulation of DUX4 activity is poorly known. We identify Matrin 3 (MATR3), whose mutation causes ALS and dominant distal myopathy, as a cellular factor controlling DUX4 expression and activity. MATR3 binds to the DUX4 DNA-binding domain and blocks DUX4-mediated gene expression, rescuing cell viability and myogenic differentiation of FSHD muscle cells, without affecting healthy muscle cells. Finally, we characterize a shorter MATR3 fragment that is necessary and sufficient to directly block DUX4-induced toxicity to the same extent as the full-length protein. Collectively, our data suggest MATR3 as a candidate for developing a treatment for FSHD.
Introduction/aims:
In our experience, patients with late-onset facioscapulohumeral muscular dystrophy type 1 (FSHD1) are frequently misdiagnosed, some for many years. The aim of this report is to document this clinical experience including the presenting symptoms and misdiagnoses and to discuss the challenges in diagnosing patients with late-onset FSHD1.
Methods:
We performed a retrospective medical record review and recorded clinical data on patients with a genetically confirmed diagnosis of FSHD1, who began to have symptoms at 50 years of age or older, and either had no family history of FSHD1 or had a history of an undiagnosed weakness in a family member.
Results:
Thirteen patients, 7 men and 6 women, met the study inclusion criteria. Age of onset ranged from 52 to 74 (mean, 59.8) years, age of diagnosis ranged from 54 to 80 (mean, 66.5) years, and duration of symptoms from onset to diagnosis was 1 to 15 (mean, 6.7) years. Prior diagnoses included lumbosacral polyradiculopathy in five (38%); statin-related myopathy in two (15%); and one each of polymyositis, inclusion-body myositis, distal myopathy, limb-girdle muscular dystrophy, unspecific myopathy, and unspecified scapular winging. For eight patients (62%), family history was suspected in deceased members or if by confirmed DNA test postdiagnosis.
Discussion:
The diagnosis of late-onset FSHD1 is often delayed by many years with patients frequently receiving misdiagnoses. FSHD1 may not be considered in the differential diagnosis of late-onset weakness due to its rarity and because its clinical features are subtler, nonspecific, and mimic other neuromuscular disorders.
Muscular dystrophies are a group of hereditary and often progressive muscle diseases. Structural or functional mutations in one of the proteins in the extracellular matrix surrounding the muscle fibre, sarcolemma, basement membrane, sarcoplasm, or nucleus can result in dystrophic diseases. According to their pathogenesis, muscular dystrophies exhibit a wide range of clinical features including, age of onset of symptoms, clinical course, pattern and severity of involvement, rate of progression, and concomitant conditions. In this chapter, the questions of what kind of complaints, physical and laboratory examination findings are detected in muscular dystrophies, and what are the genetic causes are answered. Differential diagnosis methods are briefly explained. Examples of patients with their muscle biopsy images, and changes observed in muscle dystrophies are presented. At the end of the chapter, under the title “all about the pathology of muscular dystrophies”, case examples, surprising cases, short clinical histories, and histopathological pictures are presented.KeywordsDuchenne muscular dystrophyBecker muscular dystrophyCongenital muscular dystrophyMyotonic dystrophyFacioscapulohumeral dystrophyLimb girdle muscular dystrophyEmery Dreifuss muscular dystrophyOculopharyngeal muscular dystrophy
Background:
Eating an adequate diet and maintaining a healthy body weight can be challenging for patients with muscular disorders (MD). Starting tube feeding can have a positive impact on nutritional status, functioning and quality of life. Guidelines on when to start tube feeding in adults with MD are lacking.
Objective:
We aim to review the scientific literature on indications to start tube feeding in adults with facioscapulohumeral dystrophy (FSHD), inclusion body myositis (IBM), muscular dystrophy type 1 (DM1), oculopharyngeal muscular dystrophy (OPMD) and congenital myopathies.
Methods:
This scoping review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping reviews (PRISMA-ScR) guidelines. Relevant studies were identified in Pubmed, Embase and Cinahl (April 2022). The medical subject headings (MeSH) and text words used were related to FSHD, IBM, DM1, OPMD or congenital myopathies and dysphagia, enteral nutrition or malnutrition.
Results:
Of 1046 unique articles, 9 case reports and 2 retrospective case series were included. Indications to start tube feeding were dysphagia, malnutrition/weight loss and respiratory infections (due to aspiration). Percutaneous endoscopic gastrostomy (PEG) tubes were used most often and complications were respiratory failure, problems with the tube itself, accidental tube removal, cutaneous symptoms, digestive symptoms, and peritonitis.
Conclusion:
Data on tube feeding in MD is scarce. Indications to start tube feeding were similar across the various MD. We call for more research in this field and suggest to include screening for dysphagia, aspiration and malnutrition in for the treatment of various MD.
Facioscapulohumeral muscular dystrophy (FSHD) is an incurable myopathy linked to the over-expression of the myotoxic transcription factor DUX4. Targeting DUX4 is the leading therapeutic approach, however, it is only detectable in 0.1–3.8% of FSHD myonuclei. How rare DUX4 drives FSHD and the optimal anti-DUX4 strategy are unclear. We combine stochastic gene expression with compartment models of cell states, building a simulation of DUX4 expression and consequences in FSHD muscle fibers. Investigating iDUX4 myoblasts, scRNAseq, and snRNAseq of FSHD muscle we estimate parameters including DUX4 mRNA degradation, transcription and translation rates, and DUX4 target gene activation rates. Our model accurately recreates the distribution of DUX4 and targets gene-positive cells seen in scRNAseq of FSHD myocytes. Importantly, we show DUX4 drives significant cell death despite expression in only 0.8% of live cells. Comparing scRNAseq of unfused FSHD myocytes to snRNAseq of fused FSHD myonuclei, we find evidence of DUX4 protein syncytial diffusion and estimate its rate via genetic algorithms. We package our model into freely available tools, to rapidly investigate the consequences of anti-DUX4 therapy.
Facioscapulohumeral muscular dystrophy (FSHD) is an incurable myopathy linked to the over-expression of the myotoxic transcription factor DUX4. Targeting DUX4 is the leading therapeutic approach, however, it is only detectable in 0.1–3.8% of FSHD myonuclei. How rare DUX4 drives FSHD and the optimal anti-DUX4 strategy are unclear. We combine stochastic gene expression with compartment models of cell states, building a simulation of DUX4 expression and consequences in FSHD muscle fibers. Investigating iDUX4 myoblasts, scRNAseq, and snRNAseq of FSHD muscle we estimate parameters including DUX4 mRNA degradation, transcription and translation rates, and DUX4 target gene activation rates. Our model accurately recreates the distribution of DUX4 and targets gene-positive cells seen in scRNAseq of FSHD myocytes. Importantly, we show DUX4 drives significant cell death despite expression in only 0.8% of live cells. Comparing scRNAseq of unfused FSHD myocytes to snRNAseq of fused FSHD myonuclei, we find evidence of DUX4 protein syncytial diffusion and estimate its rate via genetic algorithms. We package our model into freely available tools, to rapidly investigate the consequences of anti-DUX4 therapy.
Facioscapulohumeral muscular dystrophy (FSHD) is an incurable myopathy linked to the over-expression of the myotoxic transcription factor DUX4. Targeting DUX4 is the leading therapeutic approach, however, it is only detectable in 0.1–3.8% of FSHD myonuclei. How rare DUX4 drives FSHD and the optimal anti-DUX4 strategy are unclear. We combine stochastic gene expression with compartment models of cell states, building a simulation of DUX4 expression and consequences in FSHD muscle fibers. Investigating iDUX4 myoblasts, scRNAseq, and snRNAseq of FSHD muscle we estimate parameters including DUX4 mRNA degradation, transcription and translation rates, and DUX4 target gene activation rates. Our model accurately recreates the distribution of DUX4 and targets gene-positive cells seen in scRNAseq of FSHD myocytes. Importantly, we show DUX4 drives significant cell death despite expression in only 0.8% of live cells. Comparing scRNAseq of unfused FSHD myocytes to snRNAseq of fused FSHD myonuclei, we find evidence of DUX4 protein syncytial diffusion and estimate its rate via genetic algorithms. We package our model into freely available tools, to rapidly investigate the consequences of anti-DUX4 therapy.
Addition by Contraction
Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common hereditary neuromuscular disorders in Western populations, affecting about 1 in 20,000 people. In most patients, the disorder is associated with contraction of a D4Z4 microsatellite repeat array on chromosome 4q, but this contraction can also occur in the absence of disease, so the underlying genetic mechanisms have remained elusive. Lemmers et al. (p. 1650 , published online 19 August; see the Perspective by Mahadevan ) now show that FSHD patients carry sequence variants that create a canonical polyadenylation signal for transcripts derived from DUX4 , a homeobox gene straddling the last D4Z4 repeat unit and the adjacent sequence. Addition of poly(A) stabilizes the DUX4 transcript, which is likely to be a contributing factor in the disease.
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder which maps to chromosome 4qter, distal to the D4S139 locus. The cosmid clone 13E, isolated in a search for homeobox genes, was subsequently mapped to 4q35, also distal to D4S139. A subclone, p13E-11, detects in normal individuals a polymorphic EcoRI fragment usually larger than 28 kilobases (kb). Surprisingly, using the same probe we detected de novo DNA rearrangements, characterized by shorter EcoRI fragments (14-28 kb), in 5 out of 6 new FSHD cases. In 10 Dutch families analysed, a specific shorter fragment between 14-28 kb cosegregates with FSHD. Both observations indicate that FSHD is caused by independent de novo DNA rearrangements in the EcoRI fragment detected by p13E-11.
Each of the various neuromuscular diseases is rare. Consequently, solid epidemiological data are not available and it is often difficult to find sufficient patients for studies. For this reason, the Dutch neuromuscular database, CRAMP (Computer Registry of All Myopathies and Polyneuropathies), was developed in 2004 by the Dutch Neuromuscular Research Support Centre, to store information on patient characteristics and diagnoses (based on Rowland and McLeod's classification) in a uniform and easily retrievable manner. Care was taken to preserve data confidentiality. It is envisaged that CRAMP will prove particularly useful for studies in which multicentre collaboration is needed to recruit a sufficiently large number of patients. More than 10,000 patients with neuromuscular diseases (4,837 female, 5,476 male) have been registered since 2004, half of whom (n=5059) have peripheral nerve disorders.
This note considers the variance estimation for population size estimators based on capture–recapture experiments. Whereas a diversity of estimators of the population size has been suggested, the question of estimating the associated variances is less frequently addressed. This note points out that the technique of conditioning can be applied here successfully which also allows us to identify sources of variation: the variance due to estimation of the model parameters and the binomial variance due to sampling n units from a population of size N. It is applied to estimators typically used in capture–recapture experiments in continuous time including the estimators of Zelterman and Chao and improves upon previously used variance estimators. In addition, knowledge of the variances associated with the estimators by Zelterman and Chao allows the suggestion of a new estimator as the weighted sum of the two. The decomposition of the variance into the two sources allows also a new understanding of how resampling techniques like the Bootstrap could be used appropriately. Finally, the sample size question for capture–recapture experiments is addressed. Since the variance of population size estimators increases with the sample size, it is suggested to use relative measures such as the observed-to-hidden ratio or the completeness of identification proportion for approaching the question of sample size choice.
Prevalence, incidence and duration of a condition or illness in a steady-state population are interrelated in such a way that two of these quantities may be used to obtain the third. Data may be collected in the most expedient manner, either as prevalence or incidence series of cases, and the results expressed as both incidence and prevalence. Information about the distribution of total durations of the condition in an incidence series of cases, or about the distribution of total durations or durations-to-date in a prevalence series is necessary in order to use these relations between prevalence and incidence of a condition or illness. The duration of a condition may be taken as one measure of the effect of the condition or illness on a population, and a "treatment effect" measured by comparing two populations may be termed the etiologic duration. Exact methods are presented for interconverting the distribution of total durations of condition among prevalence and incidence series of cases arising from the same steady-state population. Both prevalence and incidence series of cases may naturally arise in the same epidemiologic study, such as the initiation and conduct of a periodic screening program, and under certain conditions the size and even the direction of the etiologic duration may differ as measured in corresponding prevalence and incidence series of cases.
Extrapolating the figures from a previous study on FSHD in a province of The Netherlands to the entire Dutch population suggests that at present a nearly complete overview is obtained of all symptomatic kindred. In 139 families, dominant inheritance was observed in 97, a pattern compatible with germline mosaicism in 6, while sporadic cases were found in 36 families. A mutation frequency of 9.6% was calculated. Mental retardation and severe retinal vasculopathy were reported in low frequencies (1%). Early onset was seen more frequently in sporadic cases. Chromosome 4 linkage appeared excluded in 3 of 22 autosomal-dominant families. The clinical pictures in the linked and nonlinked families were identical.
The results of the analysis of data for selected neurologic diseases from surveys and from several sources of mortality data were reviewed. The results of a survey in the population of Rochester, Minnesota, were presented in some detail, and the data from this source and others which are available were used in many instances to obtain estimates of incidence and prevalence for the population of the United States. The uses of such estimates for base-line comparative studies and for matters pertaining to medical care and research resources were considered. The interrelationship of genetics and epidemiology was discussed and examples of the combined effect of exogenous and hereditary factors in the causation of disease have been presented. The concept of a continuum of hereditary diseases of the nervous system was reiterated, and the need for elucidation of the biochemical nature of the genetic lesions was illustrated.
Available at: http://statline. cbs.nl/StatWeb
- Statistics Netherlands
- Statline
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Netherlands 2001–2010 [online]. Available at: http://statline.
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Statline: residual life expectancy in the Netherlands Available at: http:// statline.cbs.nl/StatWeb/publication/default
Statistics Netherlands. Statline: residual life expectancy in
the Netherlands 2001–2010 [online]. Available at: http://
statline.cbs.nl/StatWeb/publication/default.aspx?DM5
SLEN&PA571950eng&D150&D25a&D35a&D45
0&D5520-29&LA5EN&HDR5T&STB5G1%2cG2%
2cG3%2cG4&VW5T. Accessed April 28, 2014.
Statline: population numbers the Netherlands
Statistics Netherlands. Statline: population numbers the
Netherlands 2001-2010 [online]. Available at: http://statline.
cbs.nl/StatWeb/publication/?DM5SLEN&PA537296
eng&D150&D2551-60&LA5EN&HDR5T&STB5G1
Available at: http:// statline.cbs.nl/StatWeb/publication/default.aspx?DM5 SLEN&PA571950eng&D150&D25a&D35a&D45 0&D5520-29&LA5EN&HDR5T&STB5G1%2cG2% 2cG3%2cG4&VW5T
Statistics Netherlands. Statline: residual life expectancy in
the Netherlands 2001-2010 [online]. Available at: http://
statline.cbs.nl/StatWeb/publication/default.aspx?DM5
SLEN&PA571950eng&D150&D25a&D35a&D45
0&D5520-29&LA5EN&HDR5T&STB5G1%2cG2%
2cG3%2cG4&VW5T. Accessed April 28, 2014.