D4Z4 Contraction Research Articles

Single-cell spatial transcriptomics reveals a dystrophic trajectory following a developmental bifurcation of myoblast cell fates in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to abnormal derepression of the transcription activator DUX4. This effect is localized to a low percentage of cells, requiring single-cell analysis. However, single-cell/nucleus RNA-seq cannot fully capture the transcriptome of multinucleated large myotubes. To circumvent these issues, we use multiplexed error-robust fluorescent in situ hybridization (MERFISH) spatial transcriptomics that allows profiling of RNA transcripts at a subcellular resolution. We simultaneously examined spatial distributions of 140 genes, including 24 direct DUX4 targets, in in vitro differentiated myotubes and unfused mononuclear cells (MNCs) of control, isogenic D4Z4 contraction mutant and FSHD patient samples, as well as the individual nuclei within them. We find myocyte nuclei segregate into two clusters defined by the expression of DUX4 target genes, which is exclusively found in patient/mutant nuclei, whereas MNCs cluster based on developmental states. Patient/mutant myotubes are found in "FSHD-hi" and "FSHD-lo" states with the former signified by high DUX4 target expression and decreased muscle gene expression. Pseudotime analyses reveal a clear bifurcation of myoblast differentiation into control and FSHD-hi myotube branches, with variable numbers of DUX4 target-expressing nuclei found in multinucleated FSHD-hi myotubes. Gene coexpression modules related to extracellular matrix and stress gene ontologies are significantly altered in patient/mutant myotubes compared with the control. We also identify distinct subpathways within the DUX4 gene network that may differentially contribute to the disease transcriptomic phenotype. Taken together, our MERFISH-based study provides effective gene network profiling of multinucleated cells and identifies FSHD-induced transcriptomic alterations during myoblast differentiation.

Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy in Patients Clinically Suspected of FSHD Using Optical Genome Mapping.

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. 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. 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%. 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.

O11 Long-read nanopore sequencing in FSHD patients reveals CpG methylation patterns including methylation gradients in contracted D4Z4 arrays

O11 Long-read nanopore sequencing in FSHD patients reveals CpG methylation patterns including methylation gradients in contracted D4Z4 arrays

Deciphering D4Z4 CpG methylation gradients in fascioscapulohumeral muscular dystrophy using nanopore sequencing.

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.

Long-read Nanopore sequencing identified D4Z4 contractions in patients with facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic muscle disorder caused by abnormal expression of the DUX4 protein, commonly resulting from a contraction of D4Z4 repeat units with the presence of a polyadenylation (polyA) signal. More than 10 units of the D4Z4 repeat, with a length of 3.3 kb per unit, are typically required to silence DUX4 expression. Consequently, molecular diagnosis of FSHD is challenging. We used Oxford Nanopore technology to perform whole-genome sequencing of seven unrelated patients with FSHD, their six unaffected parents, and 10 unaffected controls. All seven patients were successfully identified to harbor one to five D4Z4 repeat units and the polyA signal, whereas none of the 16 unaffected individuals met the molecular diagnostic criteria. Our newly developed method provides a straightforward and powerful molecular diagnostic tool for FSHD.

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

BackgroundFacioscapulohumeral 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.MethodsWe 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.ResultsWe 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.ConclusionsOur 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.

Orofacial Muscle Weakening in Facioscapulohumeral Muscular Dystrophy (FSHD) Patients.

Background: Facioscapulohumeral muscular dystrophy is the third most commonly found type of muscular dystrophy. The aim of this study was to correlate the D4Z4 repeat array fragment size to the orofacial muscle weakening exhibited in a group of patients with a genetically supported diagnosis of FSHD. Methods: Molecular genetic analysis was performed for 52 patients (27 female and 25 male) from a group that consisted of 36 patients with autosomal dominant pedigrees and 16 patients with either sporadic or unknown family status. The patients were tested with the southern blotting technique, using EcoRI/Avrll double digestion, and fragments were detected by a p13E-11 telomeric probe. Spearman’s correlation was used to compare the fragment size with the degree of muscle weakening found in the forehead, periocular and perioral muscles. Results: A positive non-significant correlation between the DNA fragment size and severity of muscle weakness was found for the forehead (r = 0.27; p = 0187), the periocular (r = 0.24; p = 0.232) and the left and right perioral (r = 0.29; p = 0.122), (r = 0.32; p = 0.085) muscles. Conclusions: Although FSHD patients exhibited a decrease in muscular activity related to the forehead, perioral, and periocular muscles the genotype–phenotype associations confirmed a weak to moderate non-significant correlation between repeat size and the severity of muscle weakness. Orofacial muscle weakening and its association with a D4Z4 contraction alone may not have the significance to serve as a prognostic biomarker, due to the weak to moderate association. Further studies with larger sample sizes are needed to determine the degree of genetic involvement in the facial growth in FSHD patients.

Prevalence and disease progression of genetically-confirmed facioscapulohumeral muscular dystrophy type 1 (FSHD1) in China between 2001 and 2020: a nationwide population-based study

SummaryBackgroundFacioscapulohumeral muscular dystrophy type 1 (FSHD1) is a rare disease, which is often underdiagnosed due to its heterogeneous presentations and complex molecular genetic basis, leading to a lack of population-based epidemiology data, especially of prevalence and disease progression.MethodsFujian Neuromedical Centre (FNMC) is a diagnosis centre for clinical-genetic FSHD in China, and the only one employing pulsed-field gel electrophoresis (PFGE)-based Southern blotting for all FSHD1 genetic tests. Three sources distributed across all six spatial zones in China, were used to obtain information regarding FSHD1 events, namely, FNMC, Genetic and Myopathy Group (branches of the Neurology Society of the Chinese Medical Association), and “FSHD-China” (an organization supported by FSHD patients). During 2001-2020, all genetically-confirmed FSHD1 from China were registered in FNMC. Follow-up was conducted in the 20-year period to obtain data on disease progression, which was mainly described in terms of independent ambulation loss.FindingsOf the 1,744 FSHD1 genetic tests (total test number 1,802) included in the analysis, 997 (57.2%) patients from 620 families were diagnosed with FSHD1. The estimated prevalence of genetically-confirmed FSHD1 in China is 0.75 per million (95% confidence interval [CI], 0.70-0.79) during 2001-2020, with 0.78 (95% CI, 0.72-0.85) in males and 0.71 (95% CI, 0.65-0.78) in females. The estimated prevalence increased from 0.22 (95% CI, 0.19-0.26) per million in 2001-2015 to 0.53 (95% CI, 0.49-0.57) per million in 2016-2020 (p < 0.001). The prevalence in Fujian province was 7.10 per million, 4.66 per million, and 2.44 per million, during 2001-2020, 2001-2015, and 2016-2020, respectively. Among the 861 symptomatic plus asymptomatic patients of the total 997 patients, the median onset age at first-ever muscle weakness was 16 years of age (range 1-81); the median number of contracted D4Z4 repeats was 5 units (range 1-9); the median 4qA-allele-specific methylation level was 41% (range 14%-69%). Of the 977 symptomatic patients followed-up during 2001-2020, 117 patients (12.0%) lost independent ambulation. The expected duration from onset of first-ever muscle weakness to onset of independent ambulation loss was 40 years. The group with loss of independent ambulation had a smaller number of contracted D4Z4 repeats (p < 0.001) and had an earlier onset age of first-ever muscle weakness (p < 0.001) compared to the group without loss of independent ambulation.InterpretationOur research captures the largest genetically-confirmed FSHD1 population worldwide, to calculate its prevalence of 0.75 per million in China from 2001 to 2020. Approximately 12.0% of symptomatic plus asymptomatic patients of FSHD1 will lose independent ambulation in 40 years from onset of first-ever muscle weakness.FundingThis work has been supported by the grants (U2005201, 81870902, N.W.) and (81974193, 81671237, Z.Q.W.) from the National Natural Science Foundation of China; Joint Funds for the Innovation of Science and Technology of Fujian Province (2018Y9082) (N.W.), and the Key Clinical Specialty Discipline Construction Program of Fujian (N.W.).

Muscle Fiber Conduction Velocity Correlates With the Age at Onset in Mild FSHD Cases.

A majority of patients with facioscapulohumeral muscular dystrophy (FSHD) report severe fatigue. The aim of this study was to explore whether fatigability during a performance task is related to the main clinical features of the disease in mildly affected patients. A total of 19 individuals with a molecular genetic-based diagnosis of FSHD (median D4Z4 deletion length of 27 kb) performed two isometric flexions of the dominant biceps brachii at 20% of their maximal voluntary contraction (MVC) for 2 min, and then at 60% MVC until exhaustion. Fatigability indices (average rectified value, mean frequency, conduction velocity, and fractal dimension) were extracted from the surface electromyogram (sEMG) signal, and their correlations with age, age at onset, disease duration, D4Z4 contraction length, perceived fatigability, and clinical disability score were analyzed. The conduction velocity during the low level contraction showed a significant negative correlation with the age at onset (p < 0.05). This finding suggest the assessment of conduction velocity at low isometric contraction intensities, as a potential useful tool to highlight differences in muscle involvement in FSHD patients.

Type 1 FSHD with 6-10 Repeated Units: Factors Underlying Severity in Index Cases and Disease Penetrance in Their Relatives Attention.

Molecular defects in type 1 facioscapulohumeral muscular dystrophy (FSHD) are caused by a heterozygous contraction of the D4Z4 repeat array from 1 to 10 repeat units (RUs) on 4q35. This study compared (1) the phenotype and severity of FSHD1 between patients carrying 6–8 vs. 9–10 RUs, (2) the amount of methylation in different D4Z4 regions between patients with FSHD1 with different clinical severity scores (CSS). This cross-sectional multicenter study was conducted to measure functional scales and for genetic analysis. Patients were classified into two categories according to RUs: Group 1, 6–8; Group 2, 9–10. Methylation analysis was performed in 27 patients. A total of 99 carriers of a contracted D4Z4 array were examined. No significant correlations between RUs and CSS (r = 0.04, p = 0.73) and any of the clinical outcome scales were observed between the two groups. Hypomethylation was significantly more pronounced in patients with high CSS (>3.5) than those with low CSS (<1.5) (in DR1 and 5P), indicating that the extent of hypomethylation might modulate disease severity. In Group 1, the disease severity is not strongly correlated with the allele size and is mostly correlated with the methylation of D4Z4 regions.

Generation of genetically matched hiPSC lines from two mosaic facioscapulohumeral dystrophy type 1 patients.

Facioscapulohumeral dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4q resulting in sporadic misexpression of the transcription factor DUX4 in skeletal muscle tissue. In ~4% of families, de novo D4Z4 contractions occur after fertilization resulting in somatic mosaicism with control and FSHD1 cell populations present within the same patient. Reprogramming of mosaic fibroblasts from two FSHD1 patients into human induced pluripotent stem cells (hiPSCs) generated genetically matched control and FSHD1 hiPSC lines. All hiPSC lines contained a normal karyotype, expressed pluripotency genes and differentiated into cells from the three germ layers.

Analysis of the 4q35 chromatin organization reveals distinct long-range interactions in patients affected with Facio-Scapulo-Humeral Dystrophy

Facio-Scapulo Humeral dystrophy (FSHD) is the third most common myopathy, affecting 1 amongst 10,000 individuals (FSHD1, OMIM #158900). This autosomal dominant pathology is associated in 95% of cases with genetic and epigenetic alterations in the subtelomeric region at the extremity of the long arm of chromosome 4 (q arm). A large proportion of the remaining 5% of cases carry a mutation in the SMCHD1 gene (FSHD2, OMIM #158901). Here, we explored the 3D organization of the 4q35 locus by three-dimensions DNA in situ fluorescent hybridization (3D-FISH) in primary fibroblasts isolated from patients and healthy donors. We found that D4Z4 contractions and/or SMCHD1 mutations impact the spatial organization of the 4q35 region and trigger changes in the expression of different genes. Changes in gene expression were corroborated in muscle biopsies suggesting that the modified chromatin landscape impelled a modulation in the level of expression of a number of genes across the 4q35 locus in FSHD. Using induced pluripotent stem cells (hIPSC), we further examined whether chromatin organization is inherited after reprogramming or acquired during differentiation and showed that folding of the 4q35 region is modified upon differentiation. These results together with previous findings highlight the role of the D4Z4 macrosatellite repeat in the topological organization of chromatin and further indicate that the D4Z4-dependent 3D structure induces transcriptional changes of 4q35 genes expression.

Sporadic DUX4 expression in FSHD myocytes is associated with incomplete repression by the PRC2 complex and gain of H3K9 acetylation on the contracted D4Z4 allele

BackgroundFacioscapulohumeral muscular dystrophy 1 (FSHD1) has an autosomal dominant pattern of inheritance and primarily affects skeletal muscle. The genetic cause of FSHD1 is contraction of the D4Z4 macrosatellite array on chromosome 4 alleles associated with a permissive haplotype causing infrequent sporadic expression of the DUX4 gene. Epigenetically, the contracted D4Z4 array has decreased cytosine methylation and an open chromatin structure. Despite these genetic and epigenetic changes, the majority of FSHD myoblasts are able to repress DUX4 transcription. In this study we hypothesized that histone modifications distinguish DUX4 expressing and non-expressing cells from the same individuals.ResultsFSHD myocytes containing the permissive 4qA haplotype with a long terminal D4Z4 unit were sorted into DUX4 expressing and non-expressing groups. We found similar CpG hypomethylation between the groups of FSHD-affected cells suggesting that CpG hypomethylation is not sufficient to trigger DUX4 expression. A survey of histone modifications present at the D4Z4 region during cell lineage commitment revealed that this region is bivalent in FSHD iPS cells with both H3K4me3 activating and H3K27me3 repressive marks present, making D4Z4 poised for DUX4 activation in pluripotent cells. After lineage commitment, the D4Z4 region becomes univalent with H3K27me3 in FSHD and non-FSHD control myoblasts and a concomitant increase in H3K4me3 in a small fraction of cells. Chromatin immunoprecipitation (ChIP) for histone modifications, chromatin modifier proteins and chromatin structural proteins on sorted FSHD myocytes revealed that activating H3K9Ac modifications were ~ fourfold higher in DUX4 expressing FSHD myocytes, while the repressive H3K27me3 modification was ~ fourfold higher at the permissive allele in DUX4 non-expressing FSHD myocytes from the same cultures. Similarly, we identified EZH2, a member of the polycomb repressive complex involved in H3K27 methylation, to be present more frequently on the permissive allele in DUX4 non-expressing FSHD myocytes.ConclusionsThese results implicate PRC2 as the complex primarily responsible for DUX4 repression in the setting of FSHD and H3K9 acetylation along with reciprocal loss of H3K27me3 as key epigenetic events that result in DUX4 expression. Future studies focused on events that trigger H3K9Ac or augment PRC2 complex activity in a small fraction of nuclei may expose additional drug targets worthy of study.

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.

Smchd1 haploinsufficiency exacerbates the phenotype of a transgenic FSHD1 mouse model.

In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat that normally comprises 8-100 units. The D4Z4 repeat has heterochromatic features and does not express DUX4 in somatic cells. Individuals with facioscapulohumeral muscular dystrophy (FSHD) have a partial failure of somatic DUX4 repression resulting in the presence of DUX4 protein in sporadic muscle nuclei. Somatic DUX4 derepression is caused by contraction of the D4Z4 repeat to 1-10 units (FSHD1) or by heterozygous mutations in genes responsible for maintaining the D4Z4 chromatin structure in a repressive state (FSHD2). One of the FSHD2 genes is the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene. SMCHD1 mutations have also been identified in FSHD1; patients carrying a contracted D4Z4 repeat and a SMCHD1 mutation are more severely affected than relatives with only a contracted repeat or a SMCHD1 mutation. To evaluate the modifier role of SMCHD1, we crossbred mice carrying a contracted D4Z4 repeat (D4Z4-2.5 mice) with mice that are haploinsufficient for Smchd1 (Smchd1MommeD1 mice). D4Z4-2.5/Smchd1MommeD1 mice presented with a significantly reduced body weight and developed skin lesions. The same skin lesions, albeit in a milder form, were also observed in D4Z4-2.5 mice, suggesting that reduced Smchd1 levels aggravate disease in the D4Z4-2.5 mouse model. Our study emphasizes the evolutionary conservation of the SMCHD1-dependent epigenetic regulation of the D4Z4 repeat array and further suggests that the D4Z4-2.5/Smchd1MommeD1 mouse model may be used to unravel the function of DUX4 in non-muscle tissues like the skin.

Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral Muscular Dystrophy is a common form of muscular dystrophy that presents clinically with progressive weakness of the facial, scapular, and humeral muscles, with later involvement of the trunk and lower extremities. While typically inherited as autosomal dominant, facioscapulohumeral muscular dystrophy (FSHD) has a complex genetic and epigenetic etiology that has only recently been well described. The most prevalent form of the disease, FSHD1, is associated with the contraction of the D4Z4 microsatellite repeat array located on a permissive 4qA chromosome. D4Z4 contraction allows epigenetic derepression of the array, and possibly the surrounding 4q35 region, allowing misexpression of the toxic DUX4 transcription factor encoded within the terminal D4Z4 repeat in skeletal muscles. The less common form of the disease, FSHD2, results from haploinsufficiency of the SMCHD1 gene in individuals carrying a permissive 4qA allele, also leading to the derepression of DUX4, further supporting a central role for DUX4. How DUX4 misexpression contributes to FSHD muscle pathology is a major focus of current investigation. Misexpression of other genes at the 4q35 locus, including FRG1 and FAT1, and unlinked genes, such as SMCHD1, has also been implicated as disease modifiers, leading to several competing disease models. In this review, we describe recent advances in understanding the pathophysiology of FSHD, including the application of MRI as a research and diagnostic tool, the genetic and epigenetic disruptions associated with the disease, and the molecular basis of FSHD. We discuss how these advances are leading to the emergence of new approaches to enable development of FSHD therapeutics. © 2017 American Physiological Society. Compr Physiol 7:1229-1279, 2017.

Remotely acting SMCHD1 gene regulatory elements: in silico prediction and identification of potential regulatory variants in patients with FSHD.

BackgroundFacioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of the D4Z4 macro-satellite repeat on chromosome 4q35 (FSHD1) or mutations in the SMCHD1 gene (FSHD2). Recent studies have shown that the clinical manifestation of FSHD1 can be modified by mutations in the SMCHD1 gene within a given family. The absence of either D4Z4 contraction or SMCHD1 mutations in a small cohort of patients suggests that the disease could also be due to disruption of gene regulation. In this study, we postulated that mutations responsible for exerting a modifier effect on FSHD might reside within remotely acting regulatory elements that have the potential to interact at a distance with their cognate gene promoter via chromatin looping. To explore this postulate, genome-wide Hi-C data were used to identify genomic fragments displaying the strongest interaction with the SMCHD1 gene. These fragments were then narrowed down to shorter regions using ENCODE and FANTOM data on transcription factor binding sites and epigenetic marks characteristic of promoters, enhancers and silencers.ResultsWe identified two regions, located respectively ~14 and ~85 kb upstream of the SMCHD1 gene, which were then sequenced in 229 FSHD/FSHD-like patients (200 with D4Z4 repeat units <11). Three heterozygous sequence variants were found ~14 kb upstream of the SMCHD1 gene. One of these variants was found to be of potential functional significance based on DNA methylation analysis. Further functional ascertainment will be required in order to establish the clinical/functional significance of the variants found.ConclusionsIn this study, we propose an improved approach to predict the possible locations of remotely acting regulatory elements that might influence the transcriptional regulation of their associated gene(s). It represents a new way to screen for disease-relevant mutations beyond the immediate vicinity of the specific disease gene. It promises to be useful for investigating disorders in which mutations could occur in remotely acting regulatory elements.Electronic supplementary materialThe online version of this article (doi:10.1186/s40246-015-0047-x) contains supplementary material, which is available to authorized users.

Molecular combing compared to Southern blot for measuring D4Z4 contractions in FSHD

We compare molecular combing to Southern blot in the analysis of the facioscapulohumeral muscular dystrophy type 1 locus (FSHD1) on chromosome 4q35-qter (chr 4q) in genomic DNA specimens sent to a clinical laboratory for FSHD testing. A de-identified set of 87 genomic DNA specimens determined by Southern blot as normal (n = 71), abnormal with D4Z4 macrosatellite repeat array contractions (n = 7), indeterminate (n = 6), borderline (n = 2), or mosaic (n = 1) was independently re-analyzed by molecular combing in a blinded fashion. The molecular combing results were identical to the Southern blot results in 75 (86%) of cases. All contractions (n = 7) and mosaics (n = 1) detected by Southern blot were confirmed by molecular combing. Of the 71 samples with normal Southern blot results, 67 (94%) had concordant molecular combing results. The four discrepancies were either mosaic (n = 2), rearranged (n = 1), or borderline by molecular combing (n = 1). All indeterminate Southern blot results (n = 6) were resolved by molecular combing as either normal (n = 4), borderline (n = 1), or rearranged (n = 1). The two borderline Southern blot results showed a D4Z4 contraction on the chr 4qA allele and a normal result by molecular combing. Molecular combing overcomes a number of technical limitations of Southern blot by providing direct visualization of D4Z4 macrosatellite repeat arrays on specific chr 4q and chr 10q alleles and more precise D4Z4 repeat sizing. This study suggests that molecular combing has superior analytical validity compared to Southern blot for determining D4Z4 contraction size, detecting mosaicism, and resolving borderline and indeterminate Southern blot results. Further studies are needed to establish the clinical validity and diagnostic accuracy of these findings in FSHD.

Molecular Combing compared to Southern Blot in Measuring D4Z4 Contractions in FSHD (P2.027)

Molecular Combing compared to Southern Blot in Measuring D4Z4 Contractions in FSHD (P2.027)

Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy.

BackgroundBoth forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease.ResultsFamily cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease.ConclusionsThe epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0072-6) contains supplementary material, which is available to authorized users.