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

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Satomi Mitsuhashi ◽  
So Nakagawa ◽  
Mahoko Takahashi Ueda ◽  
Tadashi Imanishi ◽  
Martin C. Frith ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Single Molecule ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Single Molecule Sequencing ◽  
D4z4 Array

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 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.

scholarly journals Role of associated alleles and hypomethylation status in the clinical expression of facioscapulohumeral muscular dystrophy

2011 ◽  
Vol 152 (39) ◽  
pp. 1576-1585 ◽  
Author(s):  
Henriett Pikó ◽  
Mária Judit Molnár ◽  
Ágnes Herczegfalvi ◽  
Péter Mayer ◽  
Veronika Karcagi
Keyword(s):  
Muscular Dystrophy ◽  
Fragment Size ◽  
Restriction Analysis ◽  
Methylation Status ◽  
Prenatal Testing ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
D4z4 Repeat ◽  
Modifying Factors ◽  
Diagnostic Panel ◽  
Total Dna

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of the D4Z4 repeat region on 4q35. In addition, epigenetic modifying factors play a role in the complex pathomechanism of the disease. Aims: Introduction of a new diagnostic panel in Hungary for the extended molecular analysis of the disease which also provides new insights into the pathomechanism. Methods: In total, DNA samples of 185 clinically diagnosed FSHD patients and 71 asymptomatic relatives were analyzed by EcoRI and BlnI restriction digestion and Southern blot technique with probe p13-E11. Further investigations of the 4q35 alleles associated with the FSHD phenotype utilized qA and qB probes and a restriction analysis of the proximal D4Z4 unit by detecting a G/C SNP and the methylation status. Results: From the patients analyzed 115 had the D4Z4 repeat contraction, whereas from 71 asymptomatic family members five harbored the pathogenic fragment size. In eight families, prenatal testing had to be offered with an outcome of four affected fetuses. Methylation test was performed in 31 genetically confirmed FSHD patients and hypomethylation status was detected in all cases. All the 115 confirmed patients had 4qA alleles with the G polymorphism. Translocation events between 4q35 and the homologous 10q26 regions were also detected. Conclusion: Molecular diagnosis of FSHD became a routine approach in Hungary thus supporting the work of the clinicians, improving quality of life and genetic counseling of the affected families. The provided results from this research suggest that FSHD is associated with complex epigenetic disease mechanisms. Orv. Hetil., 2011, 152, 1576–1585.

scholarly journals Single-molecule optical mapping enables quantitative measurement of D4Z4 repeats in facioscapulohumeral muscular dystrophy (FSHD)

2019 ◽  
Vol 57 (2) ◽  
pp. 109-120 ◽  
Author(s):  
Yi Dai ◽  
Pidong Li ◽  
Zhiqiang Wang ◽  
Fan Liang ◽  
Fan Yang ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Single Molecule ◽  
Quantitative Measurement ◽  
Optical Mapping ◽  
Small Sample Size ◽  
Human Subjects ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Small Sample ◽  
Segmental Duplications ◽  
Control Subjects

PurposeFacioscapulohumeral muscular dystrophy (FSHD) is a common adult muscular dystrophy. Over 95% of FSHD cases are associated with contraction of the D4Z4 tandem repeat (~3.3 kb per unit) at 4q35 with a specific genomic configuration (haplotype) called 4qA. Molecular diagnosis of FSHD typically requires pulsed-field gel electrophoresis with Southern blotting. We aim to develop novel genomic and computational methods for characterising D4Z4 repeat numbers in FSHD.MethodsWe leveraged a single-molecule optical mapping platform that maps locations of restriction enzyme sites on high molecular weight (>150 kb) DNA molecules. We developed bioinformatics methods to address several challenges, including the differentiation of 4qA with 4qB alleles, the differentiation of 4q35 and 10q26 segmental duplications, the quantification of repeat numbers with different enzymes that may or may not have recognition sites within D4Z4 repeats. We evaluated the method on 25 human subjects (13 patients, 3 individual control subjects, 9 control subjects from 3 families) labelled by the Nb.BssSI and/or Nt.BspQI enzymes.ResultsWe demonstrated that the method gave a direct quantitative measurement of repeat numbers on D4Z4 repeats with 4qA allelic configuration and the levels of postzygotic mosaicism. Our method had high concordance with Southern blots from several cohorts on two platforms (Bionano Saphyr and Bionano Irys), but with improved quantification of repeat numbers.ConclusionWhile the study is limited by small sample size, our results demonstrated that single-molecule optical mapping is a viable approach for more refined analysis on genotype-phenotype relationships in FSHD, especially when postzygotic mosaicism is present.

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