D4Z4 Repeat Research Articles

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.

Facioscapulohumeral Disease as a myodevelopmental disease: Applying Ockham's razor to its various features.

Facioscapulohumeral muscular dystrophy (FSHD) is an exclusively human neuromuscular disease. In the last decades the cause of FSHD was identified: the loss of epigenetic repression of the D4Z4 repeat on chromosome 4q35 resulting in inappropriate transcription of DUX4. This is a consequence of a reduction of the array below 11 units (FSHD1) or of a mutation in methylating enzymes (FSHD2). Both require the presence of a 4qA allele and a specific centromeric SSLP haplotype. Muscles become involved in a rostro-caudally order with an extremely variable progression rate. Mild disease and non-penetrance in families with affected individuals is common. Furthermore, 2% of the Caucasian population carries the pathological haplotype without clinical features of FSHD.In order to explain the various features of FSHD we applied Ockham's Razor to all possible scenarios and removed unnecessary complexities. We postulate that early in embryogenesis a few cells escape epigenetic silencing of the D4Z4 repeat. Their number is assumed to be roughly inversely related to the residual D4Z4 repeat size. By asymmetric cell division, they produce a rostro-caudal and medio-lateral decreasing gradient of weakly D4Z4-repressed mesenchymal stem cells. The gradient tapers towards an end as each cell-division allows renewed epigenetic silencing. Over time, this spatial gradient translates into a temporal gradient based on a decreasing number of weakly silenced stem cells. These cells contribute to a mildly abnormal myofibrillar structure of the fetal muscles. They also form a downward tapering gradient of epigenetically weakly repressed satellite cells. When activated by mechanical trauma, these satellite cells de-differentiate and express DUX4. When fused to myofibrils they contribute to muscle cell death in various ways. Over time and dependent on how far the gradient reaches the FSHD phenotype becomes progressively manifest. We thus hypothesize FSHD to be a myodevelopmental disease with a lifelong attempt to restore DUX4 repression.

P476: Identification and accurate sizing of D4Z4 repeat units in patients suspected of facioscapulohumeral muscular dystrophy (FSHD) using optical genome mapping*

P476: Identification and accurate sizing of D4Z4 repeat units in patients suspected of facioscapulohumeral muscular dystrophy (FSHD) using optical genome mapping*

Testing for Facioscapulohumeral Muscular Dystrophy with Optical Genome Mapping.

The introduction of optical genome mapping has improved time constraints and a lack of specificity from previous methodologies when performing genome-wide analyses of samples. Optical genome mapping allows for the detection of structural variations, aberrations, and functionality traits from a single stained molecule of DNA. Though the preparation time is increased compared to previously utilized visualization techniques, optical genome mapping significantly reduces the time needed for analysis. Specifically, individual disease pipelines have been developed to rapidly analyze prepared samples. One of these diseases, Facioscapulohumeral Muscular Dystrophy (FSHD), is detected through quantification of the D4Z4 repeat array on chromosome 4q35. Optical genome mapping, with the ability to enumerate the repeats of the D4Z4 array, has demonstrated the capability to precisely diagnose FSHD. In this protocol, the preparation of samples and subsequent loading and analysis in an optical genome mapping system is discussed for the detection and analysis of FSHD. These methods should prove highly useful in FSHD analyses and beyond with the development of further disease analysis pipelines within the instrument. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Genomic DNA isolation, labeling, and staining Basic Protocol 2: Mapping and analysis with the Bionano Saphyr® system.

Long-read sequencing improves diagnostic rate in neuromuscular disorders.

Massive parallel sequencing methods, such as exome, genome, and targeted DNA sequencing, have aided molecular diagnosis of genetic diseases in the last 20 years. However, short-read sequencing methods still have several limitations, such inaccurate genome assembly, the inability to detect large structural variants, and variants located in hard-to-sequence regions like highly repetitive areas. The recently emerged PacBio single-molecule real-time (SMRT) and Oxford nanopore technology (ONT) long-read sequencing (LRS) methods have been shown to overcome most of these technical issues, leading to an increase in diagnostic rate. LRS methods are contributing to the detection of repeat expansions in novel disease-causing genes (e.g., ABCD3, NOTCH2NLC and RILPL1 causing an Oculopharyngodistal myopathy or PLIN4 causing a Myopathy with rimmed ubiquitin-positive autophagic vacuolation), of structural variants (e.g., in DMD), and of single nucleotide variants in repetitive regions (TTN and NEB). Moreover, these methods have simplified the characterization of the D4Z4 repeats in DUX4, facilitating the diagnosis of Facioscapulohumeral muscular dystrophy (FSHD). We review recent studies that have used either ONT or PacBio SMRT sequencing methods and discuss different types of variants that have been detected using these approaches in individuals with neuromuscular disorders.

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.

A feasibility study of noninvasive prenatal diagnosis in facioscapulohumeral muscular dystrophy type 1 in a Chinese family.

Objective: To demonstrate the feasibility of haplotype-based noninvasive prenatal diagnosis of Facioscapulohumeral Muscular Dystrophy type 1 (FSHD1). Methods: Bionano optical mapping was used to identify the D4Z4 structural variation of the genomic DNA sample from the proband affected with FSHD1. In addition, based on the technique of next generation sequencing, the pathogenic haplotype was determined by using trio strategy through genotyping his parents, and also fetal inheritance of paternal haplotypes was then deduced using the Hidden Markov Model. Results: Bionano optical mapping analysis revealed that the proband has only three D4Z4 repeats left in the 4q35 chromosomal region and a disease-permitting 4qA haplotype. The other normal allele of the proband contains 29 D4Z4 repeats and also a 4qA haplotype. The noninvasive cell-free fetal DNA (cffDNA)-based haplotype analysis suggested that the fetus inherited the pathogenic allele from his father and thus was predicted to be affected by FSHD1. In addition, Bionano optical mapping also demonstrated the presence of the pathogenic allele in the fetus by interrogating the genomic DNA from the amniotic fluid cells. Conclusion: Our study showed the cffDNA-based haplotyping was feasible for the noninvasive prenatal diagnosis of FSHD1, which is able to provide earlier testing results with a lower risk of miscarriage and infection than invasive techniques.

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 3.3kb 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.

Methylation of the 4q35 D4Z4 repeat defines disease status in facioscapulohumeral muscular dystrophy.

Genetic diagnosis of facioscapulohumeral muscular dystrophy (FSHD) remains a challenge in clinical practice as it cannot be detected by standard sequencing methods despite being the third most common muscular dystrophy. The conventional diagnostic strategy addresses the known genetic parameters of FSHD: the required presence of a permissive haplotype, a size reduction of the D4Z4 repeat of chromosome 4q35 (defining FSHD1) or a pathogenic variant in an epigenetic suppressor gene (consistent with FSHD2). Incomplete penetrance and epistatic effects of the underlying genetic parameters as well as epigenetic parameters (D4Z4 methylation) pose challenges to diagnostic accuracy and hinder prediction of clinical severity. In order to circumvent the known limitations of conventional diagnostics and to complement genetic parameters with epigenetic ones, we developed and validated a multistage diagnostic workflow that consists of a haplotype analysis and a high-throughput methylation profile analysis (FSHD-MPA). FSHD-MPA determines the average global methylation level of the D4Z4 repeat array as well as the regional methylation of the most distal repeat unit by combining bisulphite conversion with next-generation sequencing and a bioinformatics pipeline and uses these as diagnostic parameters. We applied the diagnostic workflow to a cohort of 148 patients and compared the epigenetic parameters based on FSHD-MPA to genetic parameters of conventional genetic testing. In addition, we studied the correlation of repeat length and methylation level within the most distal repeat unit with age-corrected clinical severity and age at disease onset in FSHD patients. The results of our study show that FSHD-MPA is a powerful tool to accurately determine the epigenetic parameters of FSHD, allowing discrimination between FSHD patients and healthy individuals, while simultaneously distinguishing FSHD1 and FSHD2. The strong correlation between methylation level and clinical severity indicates that the methylation level determined by FSHD-MPA accounts for differences in disease severity among individuals with similar genetic parameters. Thus, our findings further confirm that epigenetic parameters rather than genetic parameters represent FSHD disease status and may serve as a valuable biomarker for disease status.

6. Optical genome mapping as a potential Tier1 test for Postnatal Chromosomal Disorders – results of multi-institutional validation study of 331 retrospective clinical samples

Optical genome mapping (OGM), an emerging next-generation cytogenetics tool enables a comprehensive analysis of genomic aberrations. Recently, OGM has been shown to identify (genome-wide) all classes of SVs including, copy number changes, deletions, duplications, balanced/unbalanced genomic rearrangements (insertions, inversions, and translocations), repeat array expansions/contractions, and absence of heterozygosity (AOH). In the same assay, a concurrent or stepwise data analysis pipeline allows for sizing pathogenic FMR1 repeat expansions (FXS) as well as D4Z4 repeat contractions (FSHD1).

Cross-sectional Neuromuscular Phenotyping Study of Patients With Arhinia With SMCHD1 Variants.

Facioscapulohumeral muscular dystrophy type 2 (FSHD2) and arhinia are 2 distinct disorders caused by pathogenic variants in the same gene: SMCHD1. The mechanism underlying this phenotypic divergence remains unclear. In this study, we characterize the neuromuscular phenotype of individuals with arhinia caused by SMCHD1 variants and analyze their complex genetic and epigenetic criteria to assess their risk for FSHD2. Eleven individuals with congenital nasal anomalies, including arhinia, nasal hypoplasia, or anosmia, underwent a neuromuscular examination, genetic testing, muscle ultrasound, and muscle MRI. Risk for FSHD2 was determined by combined genetic and epigenetic analysis of 4q35 haplotype, D4Z4 repeat length, and methylation profile. We also compared expression levels of pathogenic DUX4 mRNA in primary myoblasts or dermal fibroblasts (upon myogenic differentiation or epigenetic transdifferentiation, respectively) in these individuals vs those with confirmed FSHD2. Among the 11 individuals with rare, pathogenic, heterozygous missense variants in exons 3-11 of SMCHD1, only a subset (n = 3/11; 1 male, 2 female; age 25-51 years) met the strict genetic and epigenetic criteria for FSHD2 (D4Z4 repeat unit length <21 in cis with a 4qA haplotype and D4Z4 methylation <30%). None of the 3 individuals had typical clinical manifestations or muscle imaging findings consistent with FSHD2. However, the patients with arhinia meeting the permissive genetic and epigenetic criteria for FSHD2 displayed some DUX4 expression in dermal fibroblasts under the epigenetic de-repression by drug treatment and in the primary myoblasts undergoing myogenic differentiation. In this cross-sectional study, we identified patients with arhinia who meet the full genetic and epigenetic criteria for FSHD2 and display the molecular hallmark of FSHD-DUX4 de-repression and expression in vitro-but who do not manifest with the typical clinicopathologic phenotype of FSHD2. The distinct dichotomy between FSHD2 and arhinia phenotypes despite an otherwise poised DUX4 locus implies the presence of novel disease-modifying factors that seem to operate as a switch, resulting in one phenotype and not the other. Identification and further understanding of these disease-modifying factors will provide valuable insight with therapeutic implications for both diseases.

Facioscapulohumeral dystrophy transcriptome signatures correlate with different stages of disease and are marked by different MRI biomarkers

With several therapeutic strategies for facioscapulohumeral muscular dystrophy (FSHD) entering clinical testing, outcome measures are becoming increasingly important. Considering the spatiotemporal nature of FSHD disease activity, clinical trials would benefit from non-invasive imaging-based biomarkers that can predict FSHD-associated transcriptome changes. This study investigated two FSHD-associated transcriptome signatures (DUX4 and PAX7 signatures) in FSHD skeletal muscle biopsies, and tested their correlation with a variety of disease-associated factors, including Ricci clinical severity score, disease duration, D4Z4 repeat size, muscle pathology scorings and functional outcome measures. It establishes that DUX4 and PAX7 signatures both show a sporadic expression pattern in FSHD-affected biopsies, possibly marking different stages of disease. This study analyzed two imaging-based biomarkers—Turbo Inversion Recovery Magnitude (TIRM) hyperintensity and fat fraction—and provides insights into their predictive power as non-invasive biomarkers for FSHD signature detection in clinical trials. Further insights in the heterogeneity of—and correlation between—imaging biomarkers and molecular biomarkers, as provided in this study, will provide important guidance to clinical trial design in FSHD. Finally, this study investigated the role of infiltrating non-muscle cell types in FSHD signature expression and detected potential distinct roles for two fibro-adipogenic progenitor subtypes in FSHD.

Reprogramming of adult human peripheral blood mononuclear cells into hiPSCs from two patients with facioscapulohumeral muscular dystrophy type 1

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophy. FSHD type 1 (FSHD1) is caused by multicopy contraction of D4Z4 repeats on chromosome 4q35. Human induced pluripotent stem cell (hiPSC) lines serve as important research models for various types of diseases in vitro. Here, we reprogrammed human peripheral blood mononuclear cells (PMBCs) into hiPSCs with episomal plasmid from two FSHD1 patients. These hiPSC lines maintained normal karyotype and exhibited typical morphology. Both of them could express pluripotency markers and differentiate into three layers. The hiPSC lines could be used for screening potential therapeutic targets and mechanism research.

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.

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.

A proteomics study identifying interactors of the FSHD2 gene product SMCHD1 reveals RUVBL1-dependent DUX4 repression

Structural Maintenance of Chromosomes Hinge Domain Containing 1 (SMCHD1) is a chromatin repressor, which is mutated in > 95% of Facioscapulohumeral dystrophy (FSHD) type 2 cases. In FSHD2, SMCHD1 mutations ultimately result in the presence of the cleavage stage transcription factor DUX4 in muscle cells due to a failure in epigenetic repression of the D4Z4 macrosatellite repeat on chromosome 4q, which contains the DUX4 locus. While binding of SMCHD1 to D4Z4 and its necessity to maintain a repressive D4Z4 chromatin structure in somatic cells are well documented, it is unclear how SMCHD1 is recruited to D4Z4, and how it exerts its repressive properties on chromatin. Here, we employ a quantitative proteomics approach to identify and characterize novel SMCHD1 interacting proteins, and assess their functionality in D4Z4 repression. We identify 28 robust SMCHD1 nuclear interactors, of which 12 are present in D4Z4 chromatin of myocytes. We demonstrate that loss of one of these SMCHD1 interacting proteins, RuvB-like 1 (RUVBL1), further derepresses DUX4 in FSHD myocytes. We also confirm the interaction of SMCHD1 with EZH inhibitory protein (EZHIP), a protein which prevents global H3K27me3 deposition by the Polycomb repressive complex PRC2, providing novel insights into the potential function of SMCHD1 in the repression of DUX4 in the early stages of embryogenesis. The SMCHD1 interactome outlined herein can thus provide further direction into research on the potential function of SMCHD1 at genomic loci where SMCHD1 is known to act, such as D4Z4 repeats, the inactive X chromosome, autosomal gene clusters, imprinted loci and telomeres.

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.).

High-resolution breakpoint junction mapping of proximally extended D4Z4 deletions in FSHD1 reveals evidence for a founder effect.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper a muscles. FSHD is caused by chromatin relaxation of the D4Z4 macrosatellite repeat, mostly by a repeat contraction, facilitating ectopic expression of DUX4 in skeletal muscle. Genetic diagnosis for FSHD is generally based on the sizing and haplotyping of the D4Z4 repeat on chromosome 4 by Southern blotting (SB), molecular combing or single-molecule optical mapping, which is usually straight forward but can be complicated by atypical rearrangements of the D4Z4 repeat. One of these rearrangements is a D4Z4 proximally extended deletion (DPED) allele, where not only the D4Z4 repeat is partially deleted, but also sequences immediately proximal to the repeat are lost, which can impede accurate diagnosis in all genetic methods. Previously, we identified several DPED alleles in FSHD and estimated the size of the proximal deletions by a complex pulsed-field gel electrophoresis and SB strategy. Here, using the next-generation sequencing, we have defined the breakpoint junctions of these DPED alleles at the base pair resolution in 12 FSHD families and 4 control individuals facilitating a PCR-based diagnosis of these DPED alleles. Our resultsshow that half of the DPED alleles are derivates of an ancient founder allele. For some DPED alleles, we found that genetic elements are deleted such as DUX4c, FRG2, DBE-T and myogenic enhancers necessitating re-evaluation of their role in FSHD pathogenesis.

Validation of Optical Genome Mapping for the Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy

The molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD) relies on detecting contractions of the unique D4Z4 repeat array at the chromosome 4q35 locus in the presence of a permissive 4q35A haplotype. Long, intact DNA molecules are required for accurate sizing of D4Z4 repeats. We validated the use of optical genome mapping to determine size and haplotype of D4Z4 alleles for FSHD analysis. The cohort included 36 unique DNA specimens from fresh blood samples or archived agarose plugs. High-molecular- weight DNA underwent sequence-specific labeling followed by separation and image analysis with data collection on the Saphyr system. D4Z4 allele sizes were calculated and haplotypes determined from the labeling patterns. Each specimen had previous diagnostic testing using restriction enzyme digests with EcoRI, EcoRI/BlnI, XapI, or HindIII, followed by pulsed field gel electrophoresis and Southern blot analysis with appropriate probes. Optical genome mapping detected 4q35 and 10q26 alleles ranging from 1 to 79 D4Z4 repeats and showed strong correlation with Southern blot allele sizing (R2=0.95) and haplotyping (133 of 134; 99.4% haplotype match). Analysis of inter-assay and intra-assay runs showed high reproducibility (0.03 to 0.94 %CV). Subsequent optical genome mapping for routine clinical testing from 315 clinical FSHD cases compared favorably with historical result trends. Optical genome mapping is an accurate and highly reproducible method for chromosomal abnormalities associated with FSHD.

Adenine base editing of the DUX4 polyadenylation signal for targeted genetic therapy in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation of the D4Z4 repeat resulting in misexpression of the D4Z4-encoded DUX4 gene in skeletal muscle. One of the key genetic requirements for the stable production of full-length DUX4 mRNA in skeletal muscle is a functional polyadenylation signal (ATTAAA) in exon three of DUX4 that is used in somatic cells. Base editors hold great promise to treat DNA lesions underlying genetic diseases through their ability to carry out specific and rapid nucleotide mutagenesis even in postmitotic cells such as skeletal muscle. In this study, we present a simple and straightforward strategy for mutagenesis of the somatic DUX4 polyadenylation signal by adenine base editing in immortalized myoblasts derived from independent FSHD-affected individuals. We show that mutating this critical cis-regulatory element results in downregulation of DUX4 mRNA and its direct transcriptional target genes. Our findings identify the somatic DUX4 polyadenylation signal as a therapeutic target and represent the first step toward clinical application of the CRISPR-Cas9 base editing platform for FSHD gene therapy.