FSHD muscle shows perturbation in fibroadipogenic progenitor cells, mitochondrial function and alternative splicing independently of inflammation.

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 categorize FSHD patients into distinct subtypes and understand pathomechanisms. Here, we further analyzed 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.

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.

Progesterone may be a regulator and B12 could be an indicator of the proximal D4Z4 repeat methylation status on 4q35ter.

Facioscapulohumeral dystrophy (FSHD) has a hypomethylation-related epigenetic background and exhibits a different course in male and female patients. The differences between males and females have been linked to the levels of sex hormones. This study is the first to investigate the possible effect of these hormones on methylation status. We hypothesized that the levels of sex-related hormones, estradiol, testosterone, progesterone, and prolactin might be associated with the methylation status of the proximal part of the D4Z4. We also investigated the effect of fT3, folic acid, and vitamin B12 levels. We collected blood from 28 FSHD patients and 28 controls. DNA was extracted from each individual for bisulfite methylation analysis and serum was separated for biochemical analysis of estradiol, testosterone, progesterone, prolactin, fT3, folic acid, and B12 analysis. Methylation analysis was specified to the DR1, 5P regions and the proximal region covering both DR1 and 5P. Methylation levels were compared between FSHD patients and controls. The correlation of methylation levels with estradiol, testosterone, progesterone, prolactin, fT3, folic acid, and B12 was investigated. We found that the 5P region and the proximal region were significantly hypomethylated in FSHD patients compared to the controls, but not the DR1 region. Male patients exhibited a significant reduction in DNA methylation compared to male controls. Older FSHD patients exhibited a notable decrease in fT3 levels and hypomethylation of the 5P region. Analyses of each CpG revealed seven hypomethylated positions that were significantly different from the control group. Two of the positions demonstrated a correlation with progesterone in the control group. With the exception of one position, the methylation levels were inversely correlated with vitamin B12 in FSHD patients. The results of our study indicate that the methylation of the proximal D4Z4 region, particularly at specific positions, may be associated with progesterone. In addition, vitamin B12 may be an indicator of hypomethylation. We suggest that examining position-specific methylations may be a useful approach for the development of epigenetic treatment modalities.

Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing.

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.

Best practice guidelines on genetic diagnostics of facioscapulohumeral muscular dystrophy: Update of the 2012 guidelines.

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.

First-trimester noninvasive prenatal diagnosis of seven facioscapulohumeral muscular dystrophy type 1 families using SNP-based amplicon sequencing: An earlier, rapid and safer way.

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.

The other face of facioscapulohumeral muscular dystrophy: Exploring orofacial weakness using muscle ultrasound.

INTRODUCTION/AIMS: One of the most distinct clinical features of facioscapulohumeral muscular dystrophy (FSHD) is facial weakness. It leads to diminished facial expression and functional impairments. Despite its clinical relevance, little else is known about orofacial muscle involvement. We therefore evaluated orofacial muscle involvement in a sizeable cohort of FSHD participants with muscle ultrasound. METHODS: Muscle ultrasound images of the following orofacial muscles were scored visually and quantitatively: depressor anguli oris (DAO), orbicularis oris (OO), buccinator, temporalis, masseter, digastric, zygomaticus major and minor bilaterally, and the geniohyoid. Reliability analyses of both visual and quantitative evaluations were performed. Ultrasound results were correlated with other measures: the FSHD clinical score, facial weakness score, and facial function scale. RESULTS: We included 107 FSHD participants (male 54%; age 52 ± 14 years), of whom 92% showed signs of facial weakness. The reliability of visual ultrasound analysis varied widely (κ 0.0-1.0). Quantitative ultrasound reliability was high (intraclass correlation analysis ≥ 0.96). The DAO, buccinator, OO, temporalis, and zygomaticus minor muscles were affected most often (15%-39%). The digastric, geniohyoid, zygomaticus major, and masseter muscles were least often affected (<5%). The ultrasound compound score correlated weakly to moderately with other outcome measures used (ρ = 0.3-0.7). DISCUSSION: This study adds to the understanding of orofacial weakness in FSHD, confirming the involvement of the muscles of facial expression in FSHD using ultrasound. We showed that orofacial muscle ultrasound is feasible and reliable when quantitatively assessed. Future studies should evaluate orofacial muscle ultrasound longitudinally, alongside clinical and patient-reported facial weakness outcome measures, to assess their potential as outcome measures.

Gastrointestinal and genitourinary symptoms in facioscapulohumeral muscular dystrophy: Prevalence and impact.

INTRODUCTION/AIMS: Anecdotally, patients with facioscapulohumeral muscular dystrophy (FSHD) describe gastrointestinal (GI) and genitourinary (GU) symptoms. We explored the prevalence of GI and GU symptoms and their impact on quality of life (QOL) in people with FSHD compared to healthy household controls. METHODS: In this descriptive, cross-sectional study, we emailed a survey exploring GI and GU symptoms to all FSHD Society patient contacts (n = 3507). We invited those with FSHD and unaffected household controls to respond. Non-parametric statistics were used to compare symptom frequency and impact of symptoms between respondents with FSHD and household controls. Within the FSHD group, symptom frequency was assessed relative to measures of disease progression (need for ambulatory or respiratory support). RESULTS: Surveys from 701 respondents (652 with FSHD) ≥18 years old were included in analysis. Those with FSHD had symptoms affecting both GI and GU systems more frequently than controls using ordinal rating of symptom frequency. Within the FSHD group, more advanced disease was associated with increased symptom frequency. QOL was negatively impacted by the GI and GU symptoms. There was no difference between groups in use of medications to treat these symptoms. DISCUSSION: Recognition and treatment of GI and GU symptoms in people with FSHD, particularly those with more advanced disease, could improve QOL. Additional investigation is required to confirm these findings and understand the physiology.

The lived experience of facioscapulohumeral muscular dystrophy: A systematic review and synthesis of the qualitative literature.

INTRODUCTION: In this review we sought to characterize the lived experience of people living with FSHD (pwFSHD) to help clinicians to orient their services to the needs of these individuals. METHODS: Five electronic databases were systematically searched for qualitative research studies containing quotations from pwFSHD. ENhancing Transparency in REporting the Synthesis of Qualitative research and Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines informed the methodology. Study quality was assessed using the Critical Appraisal Skills Programme Checklist tool, which measures the methodological quality of qualitative research. Data extracted from included studies were analyzed using thematic synthesis. RESULTS: Ninety-nine pwFSHD took part in the six studies included in this review - from research teams based in two countries. Five descriptive themes emerged: "Engaging with life as symptoms progress"; "The emotional journey"; "A family burden to bear"; "Social connection and disconnection"; and "Tension between visibility and invisibility." From these, two analytical themes were derived: "The emotional challenge of continuing and intensifying adaptation" and "The relational burden of rare disease." DISCUSSION: The lived experience of pwFSHD is characterized by physical, emotional, and social challenges that impact on engagement with life, particularly as symptoms progress. Further research is needed to provide a fuller understanding of the experience of pain in FSHD and of the lived experience of FSHD across cultures.

Muscle diffusion tensor imaging in facioscapulohumeral muscular dystrophy.

INTRODUCTION/AIMS: Muscle diffusion tensor imaging has not yet been explored in facioscapulohumeral muscular dystrophy (FSHD). We assessed diffusivity parameters in FSHD subjects compared with healthy controls (HCs), with regard to their ability to precede any fat replacement or edema. METHODS: Fat fraction (FF), water T2 (wT2), mean, radial, axial diffusivity (MD, RD, AD), and fractional anisotropy (FA) of thigh muscles were calculated in 10 FSHD subjects and 15 HCs. All parameters were compared between FSHD and controls, also exploring their gradient along the main axis of the muscle. Diffusivity parameters were tested in a subgroup analysis as predictors of disease involvement in muscle compartments with different degrees of FF and wT2 and were also correlated with clinical severity scores. RESULTS: We found that MD, RD, and AD were significantly lower in FSHD subjects than in controls, whereas we failed to find a difference for FA. In contrast, we found a significant positive correlation between FF and FA and a negative correlation between MD, RD, and AD and FF. No correlation was found with wT2. In our subgroup analysis we found that muscle compartments with no significant fat replacement or edema (FF < 10% and wT2 < 41 ms) showed a reduced AD and FA compared with controls. Less involved compartments showed different diffusivity parameters than more involved compartments. DISCUSSION: Our exploratory study was able to demonstrate diffusivity parameter abnormalities even in muscles with no significant fat replacement or edema. Larger cohorts are needed to confirm these preliminary findings.

Oligonucleotide Therapies for Facioscapulohumeral Muscular Dystrophy: Current Preclinical Landscape.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy, characterized by progressive and asymmetric muscle atrophy, primarily affecting muscles of the face, shoulder girdle, and upper arms before affecting muscles of the lower extremities with age and greater disease severity. FSHD is a disabling condition, and patients may also present with various extramuscular symptoms. FSHD is caused by the aberrant expression of double homeobox 4 (DUX4) in skeletal muscle, arising from compromised epigenetic repression of the D4Z4 array. DUX4 encodes the DUX4 protein, a transcription factor that activates myotoxic gene programs to produce the FSHD pathology. Therefore, sequence-specific oligonucleotides aimed at reducing DUX4 levels in patients is a compelling therapeutic approach, and one that has received considerable research interest over the last decade. This review aims to describe the current preclinical landscape of oligonucleotide therapies for FSHD. This includes outlining the mechanism of action of each therapy and summarizing the preclinical results obtained regarding their efficacy in cellular and/or murine disease models. The scope of this review is limited to oligonucleotide-based therapies that inhibit the DUX4 gene, mRNA, or protein in a way that does not involve gene editing.

De novo variants and recombination at 4q35: Hints for preimplantation genetic testing in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) has been associated with the deletion of an integral number of 3.3 kb units of the polymorphic D4Z4 repeat array at 4q35. The prenatal identification of this defect can be carried out on chorionic villi or amniocytes, whereas preimplantation genetic testing for monogenic disorders (PGT-M) requires molecular markers linked to the D4Z4 allele of reduced size. In this context the reliability of this association is crucial. To test the informativeness of the nearby polymorphic markers we investigated recombination at 4q35 using the polymorphic markers D4S1523, D4S163 and D4S139 positioned at 0.55, 0.5 and 0.21 Mb proximal to the D4Z4 array respectively. We determined the probability of recombination events to occur in the D4Z4-D4S1523 interval considering 86 subjects belonging to 12 FSHD families and found a recombination frequency of 14% between D4Z4 and D4S1523. Our study also revealed the occurrence of de novo variants and germline mosaicism. These findings highlight the recombinogenic nature of the 4q subtelomere and indicate that caution should be taken when interpreting PGT-M results. It is advisable that a woman who underwent a PGT-M cycle undertakes a prenatal DNA analysis to confirm the size of the D4Z4 alleles carried by the fetus.

The Facioscapulohumeral Muscular Dystrophy-Health Index: Development and evaluation of a disease-specific outcome measure.

INTRODUCTION/AIMS: As promising therapeutic interventions are tested among patients with facioscapulohumeral muscular dystrophy (FSHD), there is a clear need for valid and reliable outcome tools to track disease progression and therapeutic gain in clinical trials and for clinical monitoring. Our aim was to develop and validate the Facioscapulohumeral Muscular Dystrophy-Health Index (FSHD-HI) as a multifaceted patient-reported outcome measure (PRO) designed to measure disease burden in adults with FSHD. METHODS: Through initial interviews with 20 individuals and a national cross-sectional study with 328 individuals with FSHD, we identified the most prevalent and impactful symptoms in FSHD. The most relevant symptoms were included in the FSHD-HI. We used patient interviews, test-retest reliability evaluation, known groups validity testing, and factor analysis to evaluate and optimize the FSHD-HI. RESULTS: The FSHD-HI contains 14 subscales that measure FSHD disease burden from the patient's perspective. Fourteen adults with FSHD participated in semistructured beta interviews and found the FSHD-HI to be clear, usable, and relevant to them. Thirty-two adults with FSHD participated in test-retest reliability assessments, which demonstrated the high reliability of the FSHD-HI total score (intraclass correlation coefficient = 0.924). The final FSHD-HI and its subscales also demonstrated a high internal consistency (Cronbach α = 0.988). DISCUSSION: The FSHD-HI provides researchers and clinicians with a reliable and valid mechanism to measure multifaceted disease burden in patients with FSHD. The FSHD-HI may facilitate quantification of therapeutic effectiveness, as demonstrated by its use as a secondary and exploratory measure in several clinical trials.

Comparison of quantitative muscle ultrasound and whole-body muscle MRI in facioscapulohumeral muscular dystrophy type 1 patients.

INTRODUCTION: Muscle ultrasound is a fast, non-invasive and cost-effective examination that can identify structural muscular changes by assessing muscle thickness and echointensity (EI) with a quantitative analysis (QMUS). To assess applicability and repeatability of QMUS, we evaluated patients with genetically confirmed facioscapulohumeral muscular dystrophy type 1 (FSHD1), comparing their muscle ultrasound characteristics with healthy controls and with those detected by MRI. We also evaluated relationships between QMUS and demographic and clinical characteristics. MATERIALS AND METHODS: Thirteen patients were included in the study. Clinical assessment included MRC sum score, FSHD score and The Comprehensive Clinical Evaluation Form (CCEF). QMUS was performed with a linear transducer scanning bilaterally pectoralis major, deltoid, rectus femoris, tibialis anterior and semimembranosus muscles in patients and healthy subjects. For each muscle, we acquired three images, which were analysed calculating muscle EI by computer-assisted grey-scale analysis. QMUS analysis was compared with semiquantitative 1.5 T muscle MRI scale. RESULTS: All muscles in FSHD patients showed a significant increased echogenicity compared to the homologous muscles in healthy subjects. Older subjects and patients with higher FSHD score presented increased muscle EI. Tibialis anterior MRC showed a significant inverse correlation with EI. Higher median EI was found in muscles with more severe MRI fat replacement. CONCLUSIONS: QMUS allows quantitative evaluation of muscle echogenicity, displaying a tight correlation with muscular alterations, clinical and MRI data. Although a confirmation on larger sample is needed, our research suggests a possible future application of QMUS in diagnosis and management of muscular disorders.

Inhibition of DUX4 expression with antisense LNA gapmers as a therapy for facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD), characterized by progressive muscle weakness and deterioration, is genetically linked to aberrant expression of DUX4 in muscle. DUX4, in its full-length form, is cytotoxic in nongermline tissues. Here, we designed locked nucleic acid (LNA) gapmer antisense oligonucleotides (AOs) to knock down DUX4 in immortalized FSHD myoblasts and the FLExDUX4 FSHD mouse model. Using a screening method capable of reliably evaluating the knockdown efficiency of LNA gapmers against endogenous DUX4 messenger RNA in vitro, we demonstrate that several designed LNA gapmers selectively and effectively reduced DUX4 expression with nearly complete knockdown. We also found potential functional benefits of AOs on muscle fusion and structure in vitro. Finally, we show that one of the LNA gapmers was taken up and induced effective silencing of DUX4 upon local treatment in vivo. The LNA gapmers developed here will help facilitate the development of FSHD therapies.

Influence of DUX4 Expression in Facioscapulohumeral Muscular Dystrophy and Possible Treatments.

Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common form of muscular dystrophy and is characterized by muscle weakness and atrophy. FSHD is caused by the altered expression of the transcription factor double homeobox 4 (DUX4), which is involved in several significantly altered pathways required for myogenesis and muscle regeneration. While DUX4 is normally silenced in the majority of somatic tissues in healthy individuals, its epigenetic de-repression has been linked to FSHD, resulting in DUX4 aberrant expression and cytotoxicity in skeletal muscle cells. Understanding how DUX4 is regulated and functions could provide useful information not only to further understand FSHD pathogenesis, but also to develop therapeutic approaches for this disorder. Therefore, this review discusses the role of DUX4 in FSHD by examining the possible molecular mechanisms underlying the disease as well as novel pharmacological strategies targeting DUX4 aberrant expression.

[Non-ischemic priapism (post-traumatic arteriovenous fistula of the right cavernous body) in the background of Landusy-Dejerine myopathy].

Landouzy-Dejerine myopathy (Facial-shoulder-shoulder myodystrophy) is a disease which causes weakness of the muscles of the shoulder girdle, back and hip muscles, which predisposes patients to an increased risk of injury and disability. The article presents a clinical observation of non-ischemic priapism, which developed as a result of perineal trauma with the formation of a fistula of the right cavernous body in a patient against the background of facial shoulder-shoulder myodystrophy. Methods for the differential diagnosis of this condition are also discussed.

The Genetics and Epigenetics of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD), a progressive myopathy that afflicts individuals of all ages, provides a powerful model of the complex interplay between genetic and epigenetic mechanisms of chromatin regulation. FSHD is caused by dysregulation of a macrosatellite repeat, either by contraction of the repeat or by mutations in silencing proteins. Both cases lead to chromatin relaxation and, in the context of a permissive allele, aberrant expression of the DUX4 gene in skeletal muscle. DUX4 is a pioneer transcription factor that activates a program of gene expression during early human development, after which its expression is silenced in most somatic cells. When misexpressed in FSHD skeletal muscle, the DUX4 program leads to accumulated muscle pathology. Epigenetic regulators of the disease locus represent particularly attractive therapeutic targets for FSHD, as many are not global modifiers of the genome, and altering their expression or activity should allow correction of the underlying defect.

Simultaneous measurement of the size and methylation of chromosome 4qA-D4Z4 repeats in facioscapulohumeral muscular dystrophy by long-read sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder characterized by asymmetric muscle wasting and weakness. FSHD can be subdivided into two types: FSHD1, caused by contraction of the D4Z4 repeat on chromosome 4q35, and FSHD2, caused by mild contraction of the D4Z4 repeat plus aberrant hypomethylation mediated by genetic variants in SMCHD1, DNMT3B, or LRIF1. Genetic diagnosis of FSHD is challenging because of the complex procedures required. METHODS: We applied Nanopore CRISPR/Cas9-targeted resequencing for the diagnosis of FSHD by simultaneous detection of D4Z4 repeat length and methylation status at nucleotide level in genetically-confirmed and suspected patients. RESULTS: We found significant hypomethylation of contracted 4q-D4Z4 repeats in FSHD1, and both 4q- and 10q-D4Z4 repeats in FSHD2. We also found that the hypomethylation in the contracted D4Z4 in FSHD1 is moderately correlated with patient phenotypes. CONCLUSIONS: Our method contributes to the development for the diagnosis of FSHD using Nanopore long-read sequencing. This finding might give insight into the mechanisms by which repeat contraction causes disease pathogenesis.

Facioscapulohumeral muscular dystrophy-Reproductive counseling, pregnancy, and delivery in a complex multigenetic disease.

Reproductive counseling in facioscapulohumeral muscular dystrophy (FSHD) can be challenging due to the complexity of its underlying genetic mechanisms and due to incomplete penetrance of the disease. Full understanding of the genetic causes and potential inheritance patterns of both distinct FSHD types is essential: FSHD1 is an autosomal dominantly inherited repeat disorder, whereas FSHD2 is a digenic disorder. This has become even more relevant now that prenatal diagnosis and preimplantation genetic diagnosis options are available for FSHD1. Pregnancy and delivery outcomes in FSHD are usually favorable, but clinicians should be aware of the risks. We aim to provide clinicians with case-based strategies for reproductive counseling in FSHD, as well as recommendations for pregnancy and delivery.