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 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.

scholarly journals Rapid prenatal diagnosis of Facioscapulohumeral Muscular Dystrophy 1 by combined Bionano optical mapping and karyomapping

2019 ◽  
Vol 40 (3) ◽  
pp. 317-323 ◽  
Author(s):  
Yuting Zheng ◽  
Lingrong Kong ◽  
Hui Xu ◽  
Yongjie Lu ◽  
Xuechao Zhao ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Muscular Dystrophy ◽  
Optical Mapping ◽  
Facioscapulohumeral Muscular Dystrophy

scholarly journals Cellular and animal models for facioscapulohumeral muscular dystrophy

2020 ◽  
Vol 13 (10) ◽  
pp. dmm046904
Author(s):  
Alec M. DeSimone ◽  
Justin Cohen ◽  
Monkol Lek ◽  
Angela Lek
Keyword(s):  
Muscular Dystrophy ◽  
Myogenic Differentiation ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Lower Limbs ◽  
Repeat Array ◽  
Drug Candidates ◽  
Cellular Models ◽  
Wide Range

ABSTRACTFacioscapulohumeral muscular dystrophy (FSHD) is one of the most common forms of muscular dystrophy and presents with weakness of the facial, scapular and humeral muscles, which frequently progresses to the lower limbs and truncal areas, causing profound disability. Myopathy results from epigenetic de-repression of the D4Z4 microsatellite repeat array on chromosome 4, which allows misexpression of the developmentally regulated DUX4 gene. DUX4 is toxic when misexpressed in skeletal muscle and disrupts several cellular pathways, including myogenic differentiation and fusion, which likely underpins pathology. DUX4 and the D4Z4 array are strongly conserved only in primates, making FSHD modeling in non-primate animals difficult. Additionally, its cytotoxicity and unusual mosaic expression pattern further complicate the generation of in vitro and in vivo models of FSHD. However, the pressing need to develop systems to test therapeutic approaches has led to the creation of multiple engineered FSHD models. Owing to the complex genetic, epigenetic and molecular factors underlying FSHD, it is difficult to engineer a system that accurately recapitulates every aspect of the human disease. Nevertheless, the past several years have seen the development of many new disease models, each with their own associated strengths that emphasize different aspects of the disease. Here, we review the wide range of FSHD models, including several in vitro cellular models, and an array of transgenic and xenograft in vivo models, with particular attention to newly developed systems and how they are being used to deepen our understanding of FSHD pathology and to test the efficacy of drug candidates.

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 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.

Sign in / Sign up

Export Citation Format

Share Document