FSHD1 Patients Research Articles

D4Z4 Hypomethylation in Human Germ Cells.

Expression of the double homeobox 4 (DUX4) transcription factor is highly regulated in early embryogenesis and is subsequently epigenetically silenced. Ectopic expression of DUX4 due to hypomethylation of the D4Z4 repeat array on permissive chromosome 4q35 alleles is associated with facioscapulohumeral muscular dystrophy (FSHD). In peripheral blood samples from 188 healthy individuals, D4Z4 methylation was highly variable, ranging from 19% to 76%, and was not affected by age. In 48 FSHD2 patients, D4Z4 methylation varied from 3% to 30%. Given that DUX4 is one of the earliest transcribed genes after fertilization, the D4Z4 array is expected to be unmethylated in mature germ cells. Deep bisulfite sequencing of 188 mainly normozoospermic sperm samples revealed an average methylation of 2.5% (range 0.3-22%). Overall, the vast majority (78%) of individual sperm cells displayed no methylation at all. In contrast, only 19 (17.5%) of 109 individual germinal vesicle (GV) oocytes displayed D4Z4 methylation <2.5%. However, it is not unexpected that immature GV oocytes which are not usable for assisted reproduction are endowed with D4Z4 (up to 74%) hypermethylation and/or abnormal (PEG3 and GTL2) imprints. Although not significant, it is interesting to note that the pregnancy rate after assisted reproduction was higher for donors of sperm samples and oocytes with <2.5% methylation.

Muscle fibrosis as a prognostic biomarker in facioscapulohumeral muscular dystrophy: a retrospective cohort study

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant epigenetic disorder with highly variable muscle involvement and disease progression. Ongoing clinical trials, aimed at counteracting muscle degeneration and disease progression in FSHD patients, increase the need for reliable biomarkers. Muscle magnetic resonance imaging (MRI) studies showed that the appearance of STIR-positive (STIR+) lesions in FSHD muscles represents an initial stage of muscle damage, preceding irreversible adipose changes. Our study aimed to investigate fibrosis, a parameter of muscle degeneration undetectable by MRI, in relation to disease activity and progression of FSHD muscles. We histologically evaluated collagen in FSHD1 patients’ (STIR+ n = 27, STIR− n = 28) and healthy volunteers’ (n = 12) muscles by picrosirius red staining. All patients (n = 55) performed muscle MRI before biopsy, 45 patients also after 1 year and 36 patients also after 2 years. Fat content (T1 signal) and oedema/inflammation (STIR signal) were evaluated at baseline and at 1- and 2-year MRI follow-up. STIR+ muscles showed significantly higher collagen compared to both STIR− (p = 0.001) and healthy muscles (p < 0.0001). STIR− muscles showed a higher collagen content compared to healthy muscles (p = 0.0194). FSHD muscles with a worsening in fatty infiltration during 1- (P = 0.007) and 2-year (P < 0.0001) MRI follow-up showed a collagen content of 3.6- and 3.7-fold higher compared to FSHD muscles with no sign of progression. Moreover, the fibrosis was significantly higher in STIR+ muscles who showed a worsening in fatty infiltration in a timeframe of 2 years compared to both STIR− (P = 0.0006) and STIR+ muscles with no sign of progression (P = 0.02). Fibrosis is a sign of muscle degeneration undetectable at MRI never deeply investigated in FSHD patients. Our data show that 23/27 of STIR+ and 12/28 STIR− muscles have a higher amount of collagen deposition compared to healthy muscles. Fibrosis is higher in FSHD muscles with a worsening in fatty infiltration thus suggesting that its evaluation with innovative non-invasive techniques could be a candidate prognostic biomarker for FSHD, to be used to stratify patients and to evaluate the efficacy of therapeutic treatments.

P310 Direct measure of D4Z4 repetition in FSHD1 patients by applying comprehensive BLAST using nanopore sequencing

P310 Direct measure of D4Z4 repetition in FSHD1 patients by applying comprehensive BLAST using nanopore sequencing

Autosomal dominant in cis D4Z4 repeat array duplication alleles in facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation facilitates inappropriate expression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded by a retrogene that is embedded within the distal region of the D4Z4 repeat array. In the European population, the D4Z4 repeat array is usually organized in a single array that ranges between 8 and 100 units. D4Z4 chromatin relaxation and DUX4 derepression in FSHD is most often caused by repeat array contraction to 1-10 units (FSHD1) or by a digenic mechanism requiring pathogenic variants in a D4Z4 chromatin repressor like SMCHD1, combined with a repeat array between 8 and 20 units (FSHD2). With a prevalence of 1.5% in the European population, in cis duplications of the D4Z4 repeat array, where two adjacent D4Z4 arrays are interrupted by a spacer sequence, are relatively common but their relationship to FSHD is not well understood. In cis duplication alleles were shown to be pathogenic in FSHD2 patients; however, there is inconsistent evidence for the necessity of an SMCHD1 mutation for disease development. To explore the pathogenic nature of these alleles we compared in cis duplication alleles in FSHD patients with or without pathogenic SMCHD1 variant. For both groups we showed duplication-allele-specific DUX4 expression. We studied these alleles in detail using pulsed-field gel electrophoresis-based Southern blotting and molecular combing, emphasizing the challenges in the characterization of these rearrangements. Nanopore sequencing was instrumental to study the composition and methylation of the duplicated D4Z4 repeat arrays and to identify the breakpoints and the spacer sequence between the arrays. By comparing the composition of the D4Z4 repeat array of in cis duplication alleles in both groups, we found that specific combinations of proximal and distal repeat array sizes determine their pathogenicity. Supported by our algorithm to predict pathogenicity, diagnostic laboratories should now be furnished to accurately interpret these in cis D4Z4 repeat array duplications, alleles that can easily be missed in routine settings.

Muscle Ultrasound in an Open-Label Study of Losmapimod in Subjects with FSHD1 (P5-8.011)

<h3>Objective:</h3> Evaluate muscle ultrasound (US) in an open-label trial of losmapimod in FSHD1 patients. <h3>Background:</h3> Natural history studies have identified US as a viable imaging biomarker for FSHD muscle progression, complementary to MRI. Losmapimod is an orally active, selective, small molecule inhibitor of p38α/β. <h3>Design/Methods:</h3> Fourteen subjects ages 18 to 65 years with genetically confirmed FSHD1, clinical severity score 2 to 4 (range 0–5) and MRI-eligible skeletal muscles for needle biopsy received open-label 15 mg twice daily losmapimod for 52 weeks with the primary objective of safety. Assessments included safety, MRI, US, clinical outcomes, and Patient Reported Outcomes. US was performed on 7 muscles bilaterally using a standardized protocol. US echointensity was expressed as a z-score relative to matched healthy controls, with abnormal being defined as &gt;2. <h3>Results:</h3> The mean (SD) change from baseline in echogenicity of all muscles was −0.17 (0.9), in the upper extremity muscles −0.32 (0.9) and lower extremities −0.13 (1.0), represented a trend towards improvement. The distribution of muscle z-scores (&lt;2, 2–4, 4–6, and &gt;6) at baseline and after 52 weeks of treatment remained the same. Echointensity correlated strongly with MFI for the biceps brachii (r=0.84, p&lt;0.01), tibialis anterior (r=0.76, p&lt;0.01), and gastrocnemius medialis (r=0.50, p&lt;0.01). Correlations between US and clinical outcomes will be presented. <h3>Conclusions:</h3> Losmapimod demonstrated stability in muscle US over 52 weeks. <b>Disclosure:</b> The institution of Mr. Kools has received research support from Fulcrum Therapeutics. The institution of Mrs. Voermans has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Dynacure. The institution of Mrs. Voermans has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for NIH. The institution of Mrs. Voermans has received personal compensation in the range of $500-$4,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Neuromuscular disorder. The institution of Karlien Mul has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Avidity Biosciences. Dr. Jiang has received personal compensation for serving as an employee of Fulcrum Therapeutics. Dr. Shoskes has received personal compensation for serving as an employee of Fulcrum Therapeutics. An immediate family member of Dr. Shoskes has received personal compensation for serving as an employee of Exact Science. Ms. Marshall has nothing to disclose. Dr. Karlsson has received personal compensation for serving as an employee of AMRA Medical.

P.144a Muscle ultrasound in an open-label study of losmapimod in subjects with FSHD1

Evaluate muscle ultrasound (US) in an open-label trial of losmapimod in FSHD1 patients. Natural history studies have identified US as a viable imaging biomarker for FSHD muscle progression, complementary to MRI. Losmapimod is an orally active, selective, small molecule inhibitor of p38α/β. Fourteen subjects ages 18 to 65 years with genetically confirmed FSHD1, clinical severity score 2 to 4 (range 0-5) and MRI-eligible skeletal muscles for needle biopsy received open-label 15 mg twice daily losmapimod for 52 weeks with the primary objective of safety. Assessments included safety, MRI, US, clinical outcomes, and patient reported outcomes. US was performed on 7 muscles bilaterally using a standardized protocol. US echointensity was expressed as a z-score relative to matched healthy controls, with abnormal being defined as >2. The mean (SD) change from baseline in echogenicity of all muscles was -0.17 (0.9), in the upper extremity muscles -0.32 (0.9) and lower extremities -0.13 (1.0), represented a trend towards improvement1. The distribution of muscle z-scores (<2, 2-4, 4-6, and >6) at baseline and after 52 weeks of treatment remained the same. Echointensity correlated strongly with MFI for the biceps brachii (r=0.84, p<0.01), tibialis anterior (r=0.76, p<0.01), and gastrocnemius medialis (r=0.50, p<0.01). Correlations between US and clinical outcomes will be presented. Losmapimod demonstrated stability in muscle US over 52 weeks.

Longitudinal Analysis Of Mobile Smartphone Application For The Assessment Of Facioscapulohumeral Muscular Dystrophy (FSHD) Gait

Facioscapulohumeral muscular dystrophy (FSHD) can be categorized as having a loss of muscular function in the torso, pelvic girdle, and legs. FSHD patient’s gait may decline over time, which could be used as a marker of disease progression. If so, this may be beneficial for potential clinical trials. PURPOSE: Evaluate gait velocity and cadence across three gait tasks using a novel smartphone application among individuals with FSHD over 6 months. METHODS: 2 (male = 1, female = 1; avg. age = 65 years) genetically confirmed FSHD1 patients completed three gait trials that required them to walk on a level surface at home 5 out of 7 days a week at two time points, 6 months apart. Patients used a custom smartphone application (FSHD Gait), installed on a smartphone (LG KG40) and secured on the lower back using a running belt, while walking. FSHD Gait application was used to take pictures of their shoes and walking environment. The first task (T1) involved walking 10 meters 12 times once each day. The second task (T2) was to walk 10 meters 6 times, with 6 trials being performed in the morning and 6 in the afternoon with a minimum of 4 hours between sessions. In the final week (T3), participants were instructed to walk for 6 minutes without any breaks. One-way ANOVAs compared the two time points while controlling for shoe type and walking surface. RESULTS: Over the course of 6 months, neither participant expressed a significant decline in gait velocity. There was no difference in gait velocity between time point one and two for T1 (PRE = 0.93 ± 0.24 m/s, POST = 0.85 ± .04 m/s; Cohen’s d = 0.27; p = 0.75), T2 (PRE = 0.93 ± 0.15 m/s, POST = 0.88 ± 0.07 m/s; Cohen’s d = 0.50, p = 0.54) or T3 (PRE = 0.94 ± 0.21 m/s, POST = 1.00 ± .09 m/s; Cohen’s d = 0.47; p = 0.63). Similarly, over the course of 6 months, neither participant expressed decline in cadence between time point one and two for T1 (PRE = 94.07 ± 4.78 steps/min, POST = 99.66 ± 6.44 steps/min; Cohen’s d = 3.36; p = 0.13), T2 (PRE = 103.71 ± 5.17 steps/min, POST = 0.85 ± 0.04 steps/min; Cohen’s d = 0.44; 0.64) or T3 (PRE = 98.74 ± .25 steps/min, POST = 86.88 ± 11.30 steps/min; Cohen’s d = 1.03; p = 0.35). CONCLUSION: FSHD Gait application can successfully collect and analyze gait for FSHD patient assessments over a 6-month period. While cadence nor velocity significantly changed, overall both metrics declined in both participants during T1 and T2.

HOX epimutations driven by maternal SMCHD1/LRIF1 haploinsufficiency trigger homeotic transformations in genetically wildtype offspring

The body plan of animals is laid out by an evolutionary-conserved HOX code which is colinearly transcribed after zygotic genome activation (ZGA). Here we report that SMCHD1, a chromatin-modifying enzyme needed for X-inactivation in mammals, is maternally required for timely HOX expression. Using zebrafish and mouse Smchd1 knockout animals, we demonstrate that Smchd1 haplo-insufficiency brings about precocious and ectopic HOX transcription during oogenesis and embryogenesis. Unexpectedly, wild-type offspring born to heterozygous knockout zebrafish smchd1 mothers exhibited patent vertebrate patterning defects. The loss of maternal Smchd1 was accompanied by HOX epi-mutations driven by aberrant DNA methylation. We further show that this regulation is mediated by Lrif1, a direct interacting partner of Smchd1, whose knockout in zebrafish phenocopies that of Smchd1. Rather than being a short-lived maternal effect, HOX mis-regulation is stably inherited through cell divisions and persists in cultured fibroblasts derived from FSHD2 patients haploinsufficient for SMCHD1. We conclude that maternal SMCHD1/LRIF1 sets up an epigenetic state in the HOX loci that can only be reset in the germline. Such an unusual inter-generational inheritance, whereby a phenotype can be one generation removed from its genotype, casts a new light on how unresolved Mendelian diseases may be interpreted.

Identification of Serum Interleukin 6 Levels as a Disease Severity Biomarker in Facioscapulohumeral Muscular Dystrophy

Background:Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common myopathies in adults, displaying a progressive, frequently asymmetric involvement of a typical muscles’ pattern. FSHD is associated with epigenetic derepression of the polymorphic D4Z4 repeat on chromosome 4q, leading to DUX4 retrogene toxic expression in skeletal muscles. Identifying biomarkers that correlate with disease severity would facilitate clinical management and assess potential FSHD therapeutics’ efficacy.Objectives:This study purpose was to analyze serum cytokines to identify potential biomarkers in a large cohort of adult patients with FSHD.Methods:We retrospectively measured the levels of 20 pro-inflammatory and regulatory cytokines in sera from 100 genetically confirmed adult FSHD1 patients. Associations between cytokine concentrations and various clinical scores were investigated. We then measured serum and muscle interleukin 6 (IL-6) levels in a validated FSHD-like mouse model, ranging in severity and DUX4 expression.Results:IL-6 was identified as the only cytokine with a concentration correlating with several clinical severity and functional scores, including Clinical Severity Score, Manual Muscle Testing sum score, Brooke and Vignos scores. Further, FSHD patients displayed overall IL-6 levels more than twice high as control, and patients with milder phenotypes exhibited lower IL-6 serum concentration than those with severe muscular weakness. Lastly, an FSHD-like mouse model analysis confirmed that IL-6 levels positively correlate with disease severity and DUX4 expression.Conclusions:Serum IL-6, therefore, shows promise as a serum biomarker of FSHD severity in a large cohort of FSHD1 adult patients.

Cochlear Dysfunction Is a Frequent Feature of Facioscapulohumeral Muscular Dystrophy Type 1 (FSHD1).

Facioscapulohumeral muscular dystrophy type 1 (FSHD) represents one of the most common forms of muscular hereditary diseases and it is characterized by a great clinical variability with the typical muscular symptoms and other clinical features, including hearing impairment. However, etiopathogenetic mechanisms of auditory dysfunction are still not completely understood and it has been suggested that it could be assigned to a cochlear alteration that is present even in those subjects with a normal pure tonal audiometry (PTA) examination. We found out the cochlear function in 26 patients with molecular diagnosis of FSHD1 and in healthy controls. All patients underwent complete neurological and audiological examinations, including FSHD clinical score, pure-tone audiometry (PTA), and otoacoustic emissions (OAEs), in particular transient evoked otoacoustic emissions (TEOAEs) and distortion product evoked otoacoustic emissions (DPOAEs). All FSHD1 patients showed significantly reduced DPOAEs and TEOAEs, bilaterally and at all frequencies, even when considering only subjects with a normal PTA or a mild muscular involvement (FSHD score ≤ 2). No correlation between OAEs and FSHD clinical score was found. Cochlear echoes represent a sensitive tool in detecting subclinical cochlear dysfunction in FSHD1 even in subjects with normal hearing and/or subtle muscle involvement. Our study is focused on the importance of evaluating the cochlear alteration through OAEs and, in particular, by performing TEOAEs and DPOAEs sequentially, to evaluate more frequent specificities of cochlear dysfunction with a wider spectrum of analysis.

A 5-year clinical follow-up study from the Italian National Registry for FSHD

BackgroundThe natural history of facioscapulohumeral muscular dystrophy (FSHD) is undefined.MethodsAn observational cohort study was conducted in 246 FSHD1 patients. We split the analysis between index cases and carrier relatives and we classified all patients using the Comprehensive Clinical Evaluation Form (CCEF). The disease progression was measured as a variation of the FSHD score performed at baseline and at the end of 5-year follow-up (ΔFSHD score).FindingsDisease worsened in 79.4% (112/141) of index cases versus 38.1% (40/105) of carrier relatives and advanced more rapidly in index cases (ΔFSHD score 2.3 versus 1.2). The 79.1% (38/48) of asymptomatic carriers remained asymptomatic. The highest ΔFSHD score (1.7) was found in subject with facial and scapular weakness at baseline (category A), whereas in subjects with incomplete phenotype (facial or scapular weakness, category B) had lower ΔFSHD score (0.6) p < 0.0001.ConclusionsThe progression of disease is different between index cases and carrier relatives and the assessment of the CCEF categories has strong prognostic effect in FSHD1 patients.

Facioscapulohumeral muscular dystrophy 1 patients participating in the UK FSHD registry can be subdivided into 4 patterns of self-reported symptoms

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant incurable skeletal muscle disease. FSHD1 constitutes 95% of cases and is linked to truncation of the D4Z4 macrosatellite at 4q35. In most cases the condition initially presents with facial and proximal weakness of the upper limbs, but over the course of the disease involves lower limb and truncal muscles. Weakness is progressive and frequently asymmetric, which is a hallmark of the disease. Here we performed an analysis of 643 FSHD1 patients in the UK FSHD patient registry, investigating factors affecting rate of onset of 5 major FSHD symptoms: facial, periscapular, foot dorsiflexor, hip girdle weakness, and hearing loss. We found shorter D4Z4 repeat length associated with accelerated onset of each symptom. Furthermore, paternal inheritance of the pathogenic allele was associated with accelerated onset of foot dorsiflexor weakness, while pregnancy and carrying multiple children to term was associated with slower onset of all muscle symptoms. Lastly, we performed clustering analysis on age of onset of the 4 muscle symptoms across 222 patients. We identified 4 clinical presentations of FSHD1. A classical presentation (74%) and 3 facial sparing phenotypes: a mild presentation (5%) with later facial and periscapular involvement, an early shoulder presentation (10%) with accelerated periscapular weakness and an early foot presentation (9%) with accelerated foot dorsiflexor weakness. The mild presentation was associated with longer D4Z4 repeat lengths, while the early foot presentation had a female bias. We note, however that symptom progression differs significantly in these 4 clinical presentations independently of D4Z4 repeat length and gender, motivating investigation of further modifiers of FSHD1 severity.

Intronic SMCHD1 variants in FSHD: testing the potential for CRISPR-Cas9 genome editing

BackgroundFacioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the DUX4 retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of DUX4 in skeletal muscle. The common...

P.306Multicentric MRI study in a cohort of FSHD2 patients: pattern definition and differences between FSHD1 and FSHD2

P.306Multicentric MRI study in a cohort of FSHD2 patients: pattern definition and differences between FSHD1 and FSHD2

EP.36PATCHS MRI score correlates with clinical severity in facioscapulohumeral muscular dystrophy

To find the involvement pattern of skeletal muscle in FSHD patients and its correlation with clinical evaluations. PATCHS MRI scans of multi-section muscles consist of bilateral 6 muscle groups including scapular girdle, humeral, pelvic girdle, thigh, calf and axial muscles, with a total of 78 muscles. PATCHS MRI scans in 30 Chinese FSHD1 patients were semi-quantitively assessed for fat infiltration and were correlated to multiple clinical outcome measures. Cluster analysis was conducted to identify the different involvement pattern of muscles. In scapular girdle and humeral muscle, the most frequently and severely involved muscles were the trapezius, serratus anterior, rhomboideus and latissimus dorsi. And in pelvic girdle and leg muscles, they were semimembranosus, biceps femoris, gluteus minimus and semitendinosus. The fat infiltration score of total skeletal muscles correlated highly with the motor function measure, FSHD clinical score, Ricci score, 6-minute walking test and manual muscle testing (correlation coefficients -0.8767, 0.7768, 0.7705, -0.7077, 0.6973, respectively). And the correlation was stronger in PATCHS MRI than in MRI of legs. Cluster analysis indicated two distinctive patterns of muscle involvement, suggesting a group of untypical FSHD patients with early and more severely involved pelvic girdle and leg muscles. We showed a strong correlation between the fat infiltration score of PATCHS MRI and clinical outcome measures. PATCHS MRI scans unfolded a better performance in capturing the disease severity and motor functioning comparing with MRI of legs. We identified a group of untypical FSHD patients with early and more severely involved pelvic girdle and leg muscles, indicating the wide spectrum of phenotype in FSHD.

4q-D4Z4 chromatin architecture regulates the transcription of muscle atrophic genes in facioscapulohumeral muscular dystrophy.

Despite increasing insights in genome structure organization, the role of DNA repetitive elements, accounting for more than two thirds of the human genome, remains elusive. Facioscapulohumeral muscular dystrophy (FSHD) is associated with deletion of D4Z4 repeat array below 11 units at 4q35.2. It is known that the deletion alters chromatin structure in cis, leading to gene up-regulation. Here we show a genome-wide role of 4q-D4Z4 array in modulating gene expression via 3D nuclear contacts. We have developed an integrated strategy of 4q-D4Z4–specific 4C-seq and chromatin segmentation analyses, showing that 4q-D4Z4 3D interactome and chromatin states of interacting genes are impaired in FSHD1 condition; in particular, genes that have lost the 4q-D4Z4 interaction and with a more active chromatin state are enriched for muscle atrophy transcriptional signature. Expression level of these genes is restored by the interaction with an ectopic 4q-D4Z4 array, suggesting that the repeat directly modulates the transcription of contacted targets. Of note, the up-regulation of atrophic genes is a common feature of several FSHD1 and FSHD2 patients, indicating that we have identified a core set of deregulated genes involved in FSHD pathophysiology.

FSHD1 and FSHD2 form a disease continuum.

To compare the clinical features of patients showing a classical phenotype of facioscapulohumeral muscular dystrophy (FSHD) with genetic and epigenetic characteristics of the FSHD1 and FSHD2 loci D4Z4 and SMCHD1. This is a national multicenter cohort study. We measured motor strength, motor function, and disease severity by manual muscle testing sumscore, Brooke and Vignos scores, clinical severity score (CSS), and age-corrected CSS, respectively. We correlated these scores with genetic (D4Z4 repeat size and haplotype; SMCHD1 variant status) and epigenetic (D4Z4 methylation) parameters. We included 103 patients: 54 men and 49 women. Among them, we identified 64 patients with FSHD1 and 20 patients with FSHD2. Seven patients had genetic and epigenetic characteristics of FSHD1 and FSHD2, all carrying repeats of 9-10 D4Z4 repeat units (RU) and a pathogenic SMCHD1 variant. In the remaining patients, FSHD was genetically excluded or remained unconfirmed. All clinically affected SMCHD1 mutation carriers had a D4Z4 repeat of 9-16 RU on a disease permissive 4qA haplotype. These patients are significantly more severely affected by all clinical scales when compared to patients with FSHD1 with upper-sized FSHD1 alleles (8-10 RU). The overlap between FSHD1 and FSHD2 patients in the 9-10 D4Z4 RU range suggests that FSHD1 and FSHD2 form a disease continuum. The previously established repeat size threshold for FSHD1 (1-10 RU) and FSHD2 (11-20 RU) needs to be reconsidered. NCT01970735.

Subclinical myocardial injury in patients with Facioscapulohumeral muscular dystrophy 1 and preserved ejection fraction \u2013 assessment by cardiovascular magnetic resonance

BackgroundFacioscapulohumeral muscular dystrophy type 1 (FSHD1) is an autosomal dominant and the third most common inherited muscle disease. Cardiac involvement is currently described in several muscular dystrophies (MD), but there are conflicting reports in FSHD1. Mostly, FSHD1 is recognized as MD with infrequent cardiac involvement, but sudden cardiac deaths are reported in single cases.The aim of this study is to investigate whether subclinical cardiac involvement in FSHD1 patients is detectable in preserved left ventricular systolic function applying cardiovascular magnetic resonance (CMR).MethodsWe prospectively included patients with genetically confirmed FSHD1 (n = 52, 48 ± 15 years) and compared them with 29 healthy age-matched controls using a 1.5 T CMR scanner. Myocardial tissue differentiation was performed qualitatively using focal fibrosis imaging (late gadolinium enhancement (LGE)), fat imaging (multi-echo sequence for fat/water-separation) and parametric T2- and T1-mapping for quantifying inflammation and diffuse fibrosis. Extracellular volume fraction was calculated. A 12-lead electrocardiogram and 24-h Holter were performed for the assessment of MD-specific Groh-criteria and arrhythmia.ResultsFocal fibrosis by LGE was present in 13 patients (25%,10 men), fat infiltration in 7 patients (13%,5 men). T2 values did not differ between FSHD1 and healthy controls. Native T1 mapping revealed significantly higher values in patients (global native myocardial T1 values basal: FSHD1: 1012 ± 26 ms vs. controls: 985 ± 28 ms, p < 0.01, medial FSHD1: 994 ± 37 ms vs. controls: 982 ± 28 ms, p = 0.028). This was also evident in regions adjacent to focal fibrosis, indicating diffuse fibrosis. Groh-criteria were positive in 1 patient. In Holter, arrhythmic events were recorded in 10/43 subjects (23%).ConclusionsPatients with FSHD1 and preserved left ventricular ejection fraction present focal and diffuse myocardial injury. Longitudinal multi-center trials are needed to define the impact of myocardial changes as well as a relation between myocardial injury and arrhythmias on long-term prognosis and therapeutic decision-making.Trial registrationISRCTN registry with study ID ISRCTN13744381.

The French National Registry of patients with Facioscapulohumeral muscular dystrophy

BackgroundFacioscapulohumeral muscular dystrophy is a rare inherited neuromuscular disease with an estimated prevalence of 1/20,000 and France therefore harbors about 3000 FSHD patients. With research progress and the development of targeted therapies, patients’ identification through registries can facilitate and improve recruitment in clinical trials and studies.ResultsThe French National Registry of FSHD patients was designed as a mixed model registry involving both patients and physicians, through self-report and clinical evaluation questionnaires respectively, to collect molecular and clinical data. Because of the limited number of patients, data quality is a major goal of the registry and various automatic data control features have been implemented in the bioinformatics system. In parallel, data are manually validated by molecular and clinical curators. Since its creation in 2013, data from 638 FSHD patients have been collected, representing about 21% of the French FSHD population. The mixed model strategy allowed to collect 59.1% of data from both patients and clinicians; 26 and 14.9% from respectively patients and clinicians only. With the identification of the FSHD1 and FSHD2 forms, specific questionnaires have been designed. Though FSHD2 patients are progressively included, FSHD1 patients still account for the majority (94.9%). The registry is compatible with the FAIR principles as data are Findable, Accessible and Interoperable. We thus used molecular standards and standardized clinical terms used by the FILNEMUS French network of reference centers for the diagnosis and follow-up of patients suffering from a rare neuromuscular disease. The implemented clinical terms mostly map to dictionaries and terminology systems such as SNOMED-CT (75% of terms), CTV3 (61.7%) and NCIt (53.3%). Because of the sensitive nature of data, they are not directly reusable and can only be accessed as aggregated data after evaluation and approval by the registry oversight committee.ConclusionsThe French National Registry of FSHD patients belongs to a national effort to develop databases, which should now interact with other initiatives to build a European and/or an international FSHD virtual registry for the benefits of patients. It is accessible at www.fshd.fr and various useful information, links, and documents, including a video, are available for patients and professionals.

Identification of Epigenetic Regulators of DUX4-fl for Targeted Therapy of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic de-repression of the disease locus, leading to pathogenic misexpression of the DUX4 gene in skeletal muscle. While the factors and pathways involved in normal repression of the FSHD locus in healthy cells have been well characterized, very little is known about those responsible for the aberrant activation of DUX4-fl in FSHD myocytes. Reasoning that DUX4-fl activators might represent useful targets for small molecule inhibition, we performed a highly targeted, candidate-based screen of epigenetic regulators in primary FSHD myocytes. We confirmed several of the strongest and most specific candidates (ASH1L, BRD2, KDM4C, and SMARCA5) in skeletal myocytes from two other unrelated FSHD1 patients, and we showed that knockdown led to reduced levels of DUX4-fl and DUX4-FL target genes, as well as altered chromatin at the D4Z4 locus. As a second mode of validation, targeting the CRISPR/dCas9-KRAB transcriptional repressor to the promoters of several candidates also led to reduced levels of DUX4-fl. Furthermore, these candidates can be repressed by different methods in skeletal myocytes without major effects on certain critical muscle genes. Our results demonstrate that expression of DUX4-fl is regulated by multiple epigenetic pathways, and they indicate viable, druggable candidates for therapeutic target development.