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

2021 ◽  
Author(s):  
Richard J L F Lemmers ◽  
Patrick J Vliet ◽  
David San Leon Granado ◽  
Nienke Stoep ◽  
Henk Buermans ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genetic Diagnosis ◽  
Ectopic Expression ◽  
Shoulder Girdle ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
D4z4 Repeat ◽  
Molecular Combing ◽  
Arm Muscles ◽  
Chromatin Relaxation ◽  
Founder Allele

Abstract Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper arm 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, 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 Southern blot strategy. Here, using 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 results show 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.

scholarly journals Homozygous nonsense variant in LRIF1 associated with facioscapulohumeral muscular dystrophy

Neurology ◽  
2020 ◽  
Vol 94 (23) ◽  
pp. e2441-e2447 ◽  
Author(s):  
Kohei Hamanaka ◽  
Darina Šikrová ◽  
Satomi Mitsuhashi ◽  
Hiroki Masuda ◽  
Yukari Sekiguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Muscular Dystrophy ◽  
Target Gene ◽  
Disease Gene ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Target Gene Expression ◽  
D4z4 Repeat ◽  
Biopsy Examination ◽  
Chromatin Relaxation

ObjectiveFacioscapulohumeral muscular dystrophy (FSHD) is a heterogenetic disorder predominantly characterized by progressive facial and scapular muscle weakness. Patients with FSHD either have a contraction of the D4Z4 repeat on chromosome 4q35 or mutations in D4Z4 chromatin modifiers SMCHD1 and DNMT3B, both causing D4Z4 chromatin relaxation and inappropriate expression of the D4Z4-encoded DUX4 gene in skeletal muscle. In this study, we tested the hypothesis whether LRIF1, a known SMCHD1 protein interactor, is a disease gene for idiopathic FSHD2.MethodsClinical examination of a patient with idiopathic FSHD2 was combined with pathologic muscle biopsy examination and with genetic, epigenetic, and molecular studies.ResultsA homozygous LRIF1 mutation was identified in a patient with a clinical phenotype consistent with FSHD. This mutation resulted in the absence of the long isoform of LRIF1 protein, D4Z4 chromatin relaxation, and DUX4 and DUX4 target gene expression in myonuclei, all molecular and epigenetic hallmarks of FSHD. In concordance, LRIF1 was shown to bind to the D4Z4 repeat, and knockdown of the LRIF1 long isoform in muscle cells results in DUX4 and DUX4 target gene expression.ConclusionLRIF1 is a bona fide disease gene for FSHD2. This study further reinforces the unifying genetic mechanism, which postulates that FSHD is caused by D4Z4 chromatin relaxation, resulting in inappropriate DUX4 expression in skeletal muscle.

scholarly journals Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy

2017 ◽  
Author(s):  
Satomi Mitsuhashi ◽  
So Nakagawa ◽  
Mahoko Takahashi Ueda ◽  
Tadashi Imanishi ◽  
Martin C Frith ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Single Molecule ◽  
Gc Content ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Repeat Sequence ◽  
Reference Sequence ◽  
Sequencing Data ◽  
Bac Clone ◽  
D4z4 Repeat ◽  
D4z4 Array

AbstractSubtelomeric macrosatellite repeats are difficult to sequence using conventional sequencing methods owing to the high similarity among repeat units and high GC content. Sequencing these repetitive regions is challenging, even with recent improvements in sequencing technologies. Among these repeats, a haplotype carrying a particular sequence and shortening of the D4Z4 array on human chromosome 4q35 causes one of the most prevalent forms of muscular dystrophy with autosomal-dominant inheritance, facioscapulohumeral muscular dystrophy (FSHD). Here, we applied a nanopore-based ultra-long read sequencer to sequence a BAC clone containing 13 D4Z4 repeats and flanking regions. We successfully obtained the whole D4Z4 repeat sequence, including the pathogenic gene DUX4 in the last D4Z4 repeat. The estimated sequence accuracy of the total repeat region was 99.8% based on a comparison with the reference sequence. Errors were typically observed between purine or between pyrimidine bases. Further, we analyzed the D4Z4 sequence from publicly available ultra-long whole human genome sequencing data obtained by nanopore sequencing. This technology may be a new tool for studying D4Z4 repeats and pathomechanism of FSHD in the future and has the potential to widen our understanding of subtelomeric regions.

scholarly journals Single-molecule optical mapping enables accurate molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD)

2018 ◽  
Author(s):  
Yi Dai ◽  
Pidong Li ◽  
Zhiqiang Wang ◽  
Fan Liang ◽  
Fan Yang ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Molecular Diagnosis ◽  
Single Molecule ◽  
Optical Mapping ◽  
Genetic Diagnosis ◽  
Turnaround Time ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Copy Number Loss ◽  
Bioinformatics Pipeline ◽  
Repeat Array

ABSTRACTFacioscapulohumeral Muscular Dystrophy (FSHD) is a common adult muscular dystrophy in which the muscles of the face, shoulder blades and upper arms are among the most affected. FSHD is the only disease in which “junk” DNA is reactivated to cause disease, and the only known repeat array-related disease where fewer repeats cause disease. More than 95% of FSHD cases are associated with copy number loss of a 3.3kb tandem repeat (D4Z4 repeat) at the subtelomeric chromosomal region 4q35, of which the pathogenic allele contains less than 10 repeats and has a specific genomic configuration called 4qA. Currently, genetic diagnosis of FSHD requires pulsed-field gel electrophoresis followed by Southern blot, which is labor-intensive, semi-quantitative and requires long turnaround time. Here, we developed a novel approach for genetic diagnosis of FSHD, by leveraging Bionano Saphyr single-molecule optical mapping platform. Using a bioinformatics pipeline developed for this assay, we found that the method gives direct quantitative measurement of repeat numbers, can differentiate 4q35 and the highly paralogous 10q26 regions, can determine the 4qA/4qB allelic configuration, and can quantitate levels of post-zygotic mosaicism. We evaluated this approach on 5 patients (including two with post-zygotic mosaicism) and 2 patients (including one with post-zygotic mosaicism) from two separate cohorts, and had complete concordance with Southern blots, but with improved quantification of repeat numbers resolved between haplotypes. We concluded that single-molecule optical mapping is a viable approach for molecular diagnosis of FSHD and may be applied in clinical diagnostic settings once more validations are performed.

scholarly journals Cardiac Involvement in Facioscapulohumeral Muscular Dystrophy (FSHD)

2021 ◽  
Vol 12 ◽  
Author(s):  
Allison Ducharme-Smith ◽  
Stefan Nicolau ◽  
C. Anwar A. Chahal ◽  
Kirstie Ducharme-Smith ◽  
Shujah Rehman ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Atrioventricular Block ◽  
Cardiac Involvement ◽  
Case Reports ◽  
Shoulder Girdle ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Reduced Ejection Fraction ◽  
Cardiac Abnormalities ◽  
Conduction Abnormality ◽  
Conduction Abnormalities

Background: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies and predominantly affects facial and shoulder girdle muscles. Previous case reports and cohort studies identified minor cardiac abnormalities in FSHD patients, but their nature and frequency remain incompletely characterized.Methods: We reviewed cardiac, neurological and genetic findings of 104 patients with genetically confirmed FSHD.Results: The most common conduction abnormality was complete (7%) or incomplete (5%) right bundle branch block (RBBB). Bifascicular block, left anterior fascicular block, complete atrioventricular block, and 2:1 atrioventricular block each occurred in 1% of patients. Atrial fibrillation or flutter were seen in 5% of patients. Eight percent of patients had heart failure with reduced ejection fraction and 25% had valvular disease. The latter included aortic stenosis in 6% (severe in 4% and moderate in 2%) and moderate aortic regurgitation in 8%. Mitral valve prolapse (MVP) was present in 9% of patients without significant mitral regurgitation. There were no significant associations between structural or conduction abnormalities and age, degree of muscle weakness, or size of the 4q deletion.Conclusions: Both structural and conduction abnormalities can occur in FSHD. The most common abnormalities are benign (RBBB and MVP), but more significant cardiac involvement was also observed. The presence of cardiac abnormalities cannot be predicted from the severity of the neurological phenotype, nor from the genotype.

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

2020 ◽  
Vol 21 (6) ◽  
pp. 2221
Author(s):  
Emmanuelle Salort-Campana ◽  
Farzad Fatehi ◽  
Sadia Beloribi-Djefaflia ◽  
Stéphane Roche ◽  
Karine Nguyen ◽  
...  
Keyword(s):  
Disease Severity ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Clinical Severity ◽  
Cross Sectional ◽  
Repeat Array ◽  
D4z4 Repeat ◽  
Severity Scores ◽  
Group 2 ◽  
Group 1

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.

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

2019 ◽  
Vol 56 (12) ◽  
pp. 828-837 ◽  
Author(s):  
Remko Goossens ◽  
Marlinde L van den Boogaard ◽  
Richard J L F Lemmers ◽  
Judit Balog ◽  
Patrick J van der Vliet ◽  
...  
Keyword(s):  
Genome Editing ◽  
Intron Retention ◽  
Wild Type ◽  
Repeat Array ◽  
D4z4 Repeat ◽  
Intronic Mutation ◽  
Whole Exome ◽  
Efficient Suppression ◽  
The Common ◽  
Chromatin Relaxation

BackgroundFacioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the DUX4 retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of DUX4 in skeletal muscle. The common form of FSHD, FSHD1, is caused by a D4Z4 repeat array contraction. The less common form, FSHD2, is generally caused by heterozygous variants in SMCHD1.MethodsWe employed whole exome sequencing combined with Sanger sequencing to screen uncharacterised FSHD2 patients for extra-exonic SMCHD1 mutations. We also used CRISPR-Cas9 genome editing to repair a pathogenic intronic SMCHD1 variant from patient myoblasts.ResultsWe identified intronic SMCHD1 variants in two FSHD families. In the first family, an intronic variant resulted in partial intron retention and inclusion of the distal 14 nucleotides of intron 13 into the transcript. In the second family, a deep intronic variant in intron 34 resulted in exonisation of 53 nucleotides of intron 34. In both families, the aberrant transcripts are predicted to be non-functional. Deleting the pseudo-exon by CRISPR-Cas9 mediated genome editing in primary and immortalised myoblasts from the index case of the second family restored wild-type SMCHD1 expression to a level that resulted in efficient suppression of DUX4.ConclusionsThe estimated intronic mutation frequency of almost 2% in FSHD2, as exemplified by the two novel intronic SMCHD1 variants identified here, emphasises the importance of screening for intronic variants in SMCHD1. Furthermore, the efficient suppression of DUX4 after restoring SMCHD1 levels by genome editing of the mutant allele provides further guidance for therapeutic strategies.

scholarly journals Early-Onset Infantile Facioscapulohumeral Muscular Dystrophy: A Timely Review

2020 ◽  
Vol 21 (20) ◽  
pp. 7783
Author(s):  
Tai-Heng Chen ◽  
Yan-Zhang Wu ◽  
Yung-Hao Tseng
Keyword(s):  
Muscular Dystrophy ◽  
Early Onset ◽  
Case Reports ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Clinical Severity ◽  
Genetic Diagnostics ◽  
Rapid Decline ◽  
Novel Treatments ◽  
D4z4 Repeat ◽  
History Of

Facioscapulohumeral muscular dystrophy (FSHD)—the worldwide third most common inherited muscular dystrophy caused by the heterozygous contraction of a 3.3 kb tandem repeat (D4Z4) on a chromosome with a 4q35 haplotype—is a progressive genetic myopathy with variable onset of symptoms, distribution of muscle weakness, and clinical severity. While much is known about the clinical course of adult FSHD, data on the early-onset infantile phenotype, especially on the progression of the disease, are relatively scarce. Contrary to the classical form, patients with infantile FSHD more often have a rapid decline in muscle wasting and systemic features with multiple extramuscular involvements. A rough correlation between the phenotypic severity of FSHD and the D4Z4 repeat size has been reported, and the majority of patients with infantile FSHD obtain a very short D4Z4 repeat length (one to three copies, EcoRI size 10–14 kb), in contrast to the classical, slowly progressive, form of FSHD (15–38 kb). With the increasing identifications of case reports and the advance in genetic diagnostics, recent studies have suggested that the infantile variant of FSHD is not a genetically separate entity but a part of the FSHD spectrum. Nevertheless, many questions about the clinical phenotype and natural history of infantile FSHD remain unanswered, limiting evidence-based clinical management. In this review, we summarize the updated research to gain insight into the clinical spectrum of infantile FSHD and raise views to improve recognition and understanding of its underlying pathomechanism, and further, to advance novel treatments and standard care methods.

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