FSHD publications
by Manuel Cabral

2013: FSHD publications ... at work


Last autors : 

	Ehrlich M (USA)
	Gabellini  D (Italy)
	Jong YJ  (Taiwan)
	Krag TO (Danmark
	Kyba M (USA)
	Ottenheijm CA (USA)
	Ricci E (Italy)
	Tawil R (USA) (x2)
America: 5 -  Europe: 3  - Africa: 0 -  Asia: 1

	Demethylation (x2)
	Rbfox1 and calpain 3
	Sarcomeric dysfunction
	Sleep Disruption
	Disease progression
	Muscle regeneration and inflammation
	Coats syndrome 

; 80(4):392-399. Epub 2013 Jan 2.
A focal domain of extreme demethylation within D4Z4 in FSHD2.
Hartweck LM, Anderson LJ, Lemmers RJ, Dandapat A, Toso EA, Dalton JC, Tawil R, Day JW, van der Maarel SM, Kyba M.

From the Department of Pediatrics and Lillehei Heart Institute (L.M.H., L.J.A., A.D., E.A.T., M.K.), Minneapolis; Academic Health Center and Wellstone Muscular Dystrophy Center (J.C.D.), Minneapolis, MN; Neuromuscular Disease Center (R.T.), University of Rochester Medical Center, Rochester, NY; Department of Neurology and Institutes of Human Genetics and Translational Neuroscience and Wellstone Muscular Dystrophy Center (J.W.D.), Minneapolis, MN; and Department of Human Genetics (R.J.L., S.M.v.d.M.), Leiden University Medical Center, Leiden, the Netherlands.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disease with an unclear genetic mechanism. Most patients have a contraction of the D4Z4 macrosatellite repeat array at 4qter, which is thought to cause partial demethylation (FSHD1) of the contracted allele. Demethylation has been surveyed at 3 restriction enzyme sites in the first repeat and only a single site across the entire array, and current models postulate that a generalized D4Z4 chromatin alteration causes FSHD. The background of normal alleles has confounded the study of epigenetic alterations; however, rare patients (FSHD2) have a form of the disease in which demethylation is global, i.e., on all D4Z4 elements throughout the genome. Our objective was to take advantage of the global nature of FSHD2 to identify where disease-relevant methylation changes occur within D4Z4.

METHODS: Using bisulfite sequencing of DNA from blood and myoblast cells, methylation levels at 74 CpG sites across 3 disparate regions within D4Z4 were measured in FSHD2 patients and controls.

RESULTS: We found that rates of demethylation caused by FSHD2 are not consistent across D4Z4. We identified a focal region of extreme demethylation within a 5' domain, which we named DR1. Other D4Z4 regions, including the DUX4 ORF, were hypomethylated but to a much lesser extent.

CONCLUSIONS: These data challenge the simple view that FSHD is caused by a broad "opening" of D4Z4 and lead us to postulate that the region of focal demethylation is the site of action of the key D4Z4 chromatin regulatory factors that go awry in FSHD


PLoS Genet. 2013 Jan; Epub 2013 Jan 3.

Rbfox1 downregulation and altered calpain 3 splicing by FRG1 in a mouse model of FSHD.
Pistoni M, Shiue L, Cline MS, Bortolanza S, Neguembor MV, Xynos A, Ares M Jr, Gabellini D.
SourceDulbecco Telethon Institute and Division of Regenerative Medicine, San Raffaele Scientific Institute, Milano, Italy.

Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6-) is increased. 

Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.

Neurology. 2013 Feb 19;80(8):733-737.

Sarcomeric dysfunction contributes to muscle weakness in fshd.

Lassche S, Stienen GJ, Irving TC, van der Maarel SM, Voermans NC, Padberg GW, Granzier H, van Engelen BG, Ottenheijm CA.

OBJECTIVE: To investigate whether sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy (FSHD).

METHODS: Sarcomeric function was evaluated by contractile studies on demembranated single muscle fibers obtained from quadriceps muscle biopsies of 4 patients with FSHD and 4 healthy controls. The sarcomere length dependency of force was determined together with measurements of thin filament length using immunofluorescence confocal scanning laser microscopy. X-ray diffraction techniques were used to study myofilament lattice spacing.

RESULTS: FSHD muscle fibers produced only 70% of active force compared to healthy controls, a reduction which was exclusive to type II muscle fibers. Changes in force were not due to changes in thin filament length or sarcomere length. Passive force was increased 5- to 12-fold in both fiber types, with increased calcium sensitivity of force generation and decreased myofilament lattice spacing, indicating compensation by the sarcomeric protein titin.

CONCLUSIONS: We have demonstrated a reduction in sarcomeric force in type II FSHD muscle fibers, and suggest compensatory mechanisms through titin stiffening. Based on these findings, we propose that sarcomeric dysfunction plays a critical role in the development of muscle weakness in FSHD.

Pain Med. 2013 Feb 6. 
Pain and the Alpha-Sleep Anomaly: A Mechanism of Sleep Disruption in Fshd.
Della Marca G, Frusciante R, Vollono C, Iannaccone E, Dittoni S, Losurdo A, Testani E, Gnoni V, Colicchio S, Di Blasi C, Erra C, Mazza S, Ricci E.
Institute of Neurology, Catholic University, Rome, Italy.

OBJECTIVE: To measure the presence of the alpha-sleep anomaly in facioscapulohumeral muscular dystrophy (FSHD) and to evaluate the association between the sleep electroencephalogram (EEG) pattern and the presence of musculoskeletal pain.
DESIGN: Cross-sectional study.
SETTING: Sleep laboratory.
SUBJECTS: Fifty-five consecutive adult FSHD patients, 26 women and 29 men, age 49.6??15.1 years (range 18-76).
INTERVENTIONS: Questionnaires and polysomnography.
OUTCOME MEASURES: Patients were asked to indicate if in the 3 months before the sleep study they presented persisting or recurring musculoskeletal pain. Patients who reported pain were asked to fill in the Italian version of the Brief Pain Inventory and the McGill Pain questionnaire, and a 101-point visual analog scale (VAS) for pain intensity. Polysomnographic recordings were performed. EEG was analyzed by means of Fast Fourier Transform. Four power spectra bands (d 0-4?Hz, ? 4-8?Hz, a 8-14?Hz,  14-32?Hz) were computed. Sleep macrostructure parameters and alpha/delta EEG power ratio during non rapid eye movement (NREM) sleep were compared between patients with and without pain.
RESULTS: Forty-two patients in our sample reported chronic pain. VAS mean score was 55.2??23.8 (range 10-100), pain rating index score was 13.8??10.2, and present pain intensity was 2.5??0.8. The statistical analysis documented an increased occurrence of the alpha and beta rhythms during NREM sleep in FSHD patients with pain. Significant correlations were observed between the alpha/delta power ratio during NREM sleep and pain measures.
CONCLUSIONS: Chronic musculoskeletal pain is frequent in FSHD patients, and it represents a major mechanism of sleep disruption.

Neuromuscul Disord. 2013 Feb 11. 
Reevaluating measures of disease progression in facioscapulohumeral muscular dystrophy.
Statland JM, McDermott MP, Heatwole C Martens WB, Pandya S, van der Kooi , Kissel JT, Wagner KR, Tawil R.

Department of Neurology, University of Rochester Medical Center, Rochester, NY, USA. Electronic address: Jeffrey_Statland@URMC.Rochester.edu.

Recent advances in the understanding of the molecular pathophysiology of facioscapulohumeral muscular dystrophy (FSHD) have identified potential therapeutic targets. Consequently, an accurate understanding of disease progression in FSHD is crucial for the design of future clinical trials. Data from 228 subjects in 3 clinical trials and 1 natural history study were compared to examine disease progression in FSHD. All studies utilized the same techniques for manual muscle testing and maximum voluntary isometric contraction testing. Both techniques yield a total strength score that can be followed over time as an indicator of disease progression. Whereas natural history data showed a decrease in strength over 1year, there was an apparent increase in strength at 6months in 2 of the 3 clinical trials in both the placebo and treatment groups, that persisted for up to 1year for maximum voluntary isometric contraction testing. Variability estimates from the clinical trial data were consistent with those seen in the natural history data. 
Patients in clinical trials in FSHD may have better outcomes than those in natural history studies, regardless of treatment assignment, emphasizing the importance of placebo groups and the need for caution when interpreting the strength results of controlled and uncontrolled trials.

Acta Neurol Scand. 2013 Feb 15. 

Muscle regeneration and inflammation in patients with facioscapulohumeral muscular dystrophy.

Hauerslev S, Orngreen MC, Hertz JM, Vissing J, Krag TO.
SourceNeuromuscular Research Unit, Department of Neurology, Rigs hospitalet, University of Copenhagen, Copenhagen, Denmark.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate whether inflammation and regeneration are prominent in mildly affected muscles of patients with facioscapulohumeral muscular dystrophy type 1A (FSHD1A). Inflammation in muscle has been suggested by MRI studies in patients with FSHD1A.

METHODS: We analysed immunohistological and histological stains of muscle biopsies from 24 patients with FSHD1A, using 10 patients with Becker muscular dystrophy (BMD) for comparison.

RESULTS: Internalized nuclei were more prevalent in BMD (23.7  10.8%) vs FSHD1A (6.3  6.8%; P < 0.001), indicating more past regenerating fibres in BMD. Recently regenerating fibres, expressing neonatal myosin heavy chain and vimentin, did not differ significantly between patients with FSHD1A (1.1  2.9%) and patients with BMD (1.8  1.9%). Regeneration was not correlated with the number of KpnI restriction fragment repeats, an FSHD1A-defining genotype property within the D4Z4 locus, or overall disease severity in patients with FSHD1A. Macrophages were more prevalent in FSHD1A (0.50  0.63 per mm(2) ) vs BMD (0.07  0.07 per mm(2) ), whereas inflammatory T cells were equally infrequent.

CONCLUSIONS: Macrophages were more prevalent in patients with FSHD1A and could be an important pathogenic mechanism for the initiation of the dystrophic process. Furthermore, regeneration was unrelated to genotype and disease severity in FSHD1A.


Epigenetics. 2013 Feb 15;8(3).
Early de novo DNA methylation and prolonged demethylation in the muscle lineage.
Tsumagari K, Baribault C, Terragni J, Varley KE, Gertz J, Pradhan S, Badoo M, Crain CM, Song L, Crawford GE, Myers RM, Lacey M, Ehrlich M.
SourceProgram in Human Genetics and Tulane Cancer Center; Tulane Health Sciences Center; New Orleans, LA USA.

Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues.

Neuromuscul Disord. 2013 Feb 20.
Infantile fshd revisited: Expansion of clinical phenotypes in patients with a very short EcoRI fragment.

Chen TH, Lai YH, Lee PL, Hsu JH, Goto K, Hayashi YK, Nishino I, Lin CW, Shih HH, Huang CC, Liang WC, Wang WF, Jong YJ.
Division of Pediatric Emergency, Department of Emergency, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.

Contrary to the classical form, infantile facioscapulohumeral muscular dystrophy (FSHD) usually denotes a severe phenotype and is frequently associated with extramuscular involvements. To elucidate the genotype-phenotype correlation in this severe subgroup, we identified a cohort of nine patients with infantile FSHD who also carried a very short (10-13kb) EcoRI fragment. Their current age ranged from 8 to 33years and age of onset ranged from 0.4 to 5years. One patient even manifested his first FSHD-related symptoms at as early as 5months of age, including inability to smile, poor response to call, and infantile spasms. To date, four patients were wheelchair-bound and six patients had asymmetric weakness. Sensorineural hearing loss and abnormal fundoscopic findings were observed in eight and all of patients respectively. Three with the smallest EcoRI fragments (10-11kb, with normal length being 50-300kb) had mental retardation. Two of these had epilepsy. Cardiac arrhythmias were found in five patients. Restrictive ventilatory defects were observed in seven patients, with one progressing to chronic respiratory failure. Two had swallowing difficulties; one of these required gastrostomy. We identified several rarely reported phenotypes in infantile FSHD, including cardiac arrhythmia, respiratory insufficiency, and swallowing difficulties. There seems to be a correlation between the severity of phenotype and the very short EcoRI fragment in the chromosome 4q35 region. We conclude that the high frequency of multi-organ involvements in this severe FSHD variant suggests the need for an early and multidisciplinary intervention.

Neurology. 2013 Feb 27. 
Coats syndrome in facioscapulohumeral dystrophy type 1: Frequency and D4Z4 contraction size.
Statland JM, Sacconi S, Farmakidis C, Donlin-Smith CM, Chung M, Tawil R.

OBJECTIVE: To investigate the frequency of Coats syndrome and its association with D4Z4 contraction size in patients with facioscapulohumeral muscular dystrophy type 1 (FSHD1).

METHODS: We searched a North American FSHD registry and the University of Rochester (UR) FSHD research database, reviewed the literature, and sent surveys to 14 FSHD referral centers in the United States and overseas to identify patients with genetically confirmed FSHD1 with a diagnosis of Coats syndrome.

RESULTS: Out of 357 genetically confirmed patients in a North American FSHD registry and 51 patients in the UR database, 3 patients had a self-reported history of Coats disease (0.8%; 95% confidence interval 0.2%-2.2%). In total, we identified 14 patients with FSHD with known genetic contraction size and Coats syndrome confirmed by ophthalmologic examination: 10 from our survey and 4 from the literature. The median age at diagnosis of Coats syndrome was 10 years (interquartile range 14 years). The median D4Z4 fragment size was 13 kilobases (kb) (interquartile range 1 kb). One patient was mosaic (55% 11 kb, and 45% 78 kb).

CONCLUSIONS: Coats syndrome is a rare extramuscular complication of FSHD1 associated with large D4Z4 contractions. Closer surveillance for retinal complications is warranted in patients with D4Z4 fragments =15 kb.