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

Hiramuki, Yosuke; Kure, Yuriko; Saito, Yoshihiko; Ogawa, Megumu; Ishikawa, Kinya; Mori‐Yoshimura, Madoka; Oya, Yasushi; Takahashi, Yūji; Kim, Dae-Seong; Arai, Noriko; Mori, Chie; Matsumura, Tsuyoshi; Hamano, Tadanori; Nakamura, Kenichiro; Ikezoe, Koji; Hayashi, Shinichiro; Goto, Yu‐ichi; Noguchi, S.; Nishino, Ichizo

doi:10.1186/s12967-022-03743-7

Cited by 18 publications

(18 citation statements)

References 25 publications

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“…Furthermore, a recent study by Hiramuki et al, 2022 tested a long-read sequencing-based approach, which allowed the authors to simultaneously analyze the D4Z4 methylation and size in FSHD patients. Additionally in this case, the authors found reduced global D4Z4 methylation levels in FSHD samples and provide precise insights into the pathological epigenetic status of D4Z4 locus [ 20 ]. Indeed, our results are consistent with all the aforementioned data and further support the applicability of DNA methylation assessment and 4q haplotyping to prioritize or exclude patients for FSHD diagnostic testing.…”

Section: Discussionmentioning

confidence: 99%

“…This finding is in accordance with previous studies reporting similar observations [ 17 , 21 ]. In addition, the application of long read sequencing found comparable reduced D4Z4 methylation levels for both 4q and 10q in FSHD2 patients [ 20 ].…”

Section: Discussionmentioning

confidence: 99%

“…Considering that the DNA methylation status representative of D4Z4 locus has been recognized as a hallmark of the disease, several research studies tested it as a possible diagnostic biomarker. In particular, a number of protocols have been proposed to assess the methylation status, although different CpG sites/regions, biological sources and variable sample sizes have been used [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Given these premises, mathylation analysis and Machine Learning (ML) pipelines were tested as possible methods to prioritize FSHD subjects for standard molecular testing and supporting the clinical diagnosis.…”

Section: Introductionmentioning

confidence: 99%

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D4Z4 Methylation Levels Combined with a Machine Learning Pipeline Highlight Single CpG Sites as Discriminating Biomarkers for FSHD Patients

Caputo

Megalizzi

Fabrizio

et al. 2022

Cells

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The study describes a protocol for methylation analysis integrated with Machine Learning (ML) algorithms developed to classify Facio-Scapulo-Humeral Dystrophy (FSHD) subjects. The DNA methylation levels of two D4Z4 regions (DR1 and DUX4-PAS) were assessed by an in-house protocol based on bisulfite sequencing and capillary electrophoresis, followed by statistical and ML analyses. The study involved two independent cohorts, namely a training group of 133 patients with clinical signs of FSHD and 150 healthy controls (CTRL) and a testing set of 27 FSHD patients and 25 CTRL. As expected, FSHD patients showed significantly reduced methylation levels compared to CTRL. We utilized single CpG sites to develop a ML pipeline able to discriminate FSHD subjects. The model identified four CpGs sites as the most relevant for the discrimination of FSHD subjects and showed high metrics values (accuracy: 0.94, sensitivity: 0.93, specificity: 0.96). Two additional models were developed to differentiate patients with lower D4Z4 size and patients who might carry pathogenic variants in FSHD genes, respectively. Overall, the present model enables an accurate classification of FSHD patients, providing additional evidence for DNA methylation as a powerful disease biomarker that could be employed for prioritizing subjects to be tested for FSHD.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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D4Z4 Methylation Levels Combined with a Machine Learning Pipeline Highlight Single CpG Sites as Discriminating Biomarkers for FSHD Patients

Caputo

Megalizzi

Fabrizio

et al. 2022

Cells

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“…Recently, Caputo and colleagues [57,58] developed a protocol for methylation analysis of specific CpG residues using iMachine Learning (ML) algorithms to classify FSHD cases. Hiramuki et al , [59 ▪ ] applied long-read sequencing through a Nanopore CRISPR/Cas9-targeted resequencing to diagnose FSHD by simultaneous detection of D4Z4 repeat length and methylation status at the nucleotide level in FSHD patients. Erdmann et al [60] developed a methylation-based diagnostic workflow comprising a haplotype and high-throughput methylation profile analyses (FSHD-MPA).…”

Section: Resultsmentioning

confidence: 99%

The FSHD jigsaw: are we placing the tiles in the right position?

Salsi,

Vattemi,

Tupler

2023

Current Opinion in Neurology

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Purpose of reviewFacioscapulohumeral muscular dystrophy (FSHD) is one of the most common myopathies, involving over 870,000 people worldwide and over 20 FSHD national registries. Our purpose was to summarize the main objectives of the scientific community on this topic and the moving trajectories of research from the past to the present. Recent findingsTo date, research is mainly oriented toward deciphering the molecular and pathogenetic basis of the disease by investigating DUX4-mediated muscle alterations. Accordingly, FSHD drug development has been escalating in the last years in an attempt to silence DUX4 or to block its downstream effectors. Breakthroughs in the field include the awareness that new biomarkers and outcome measures are required for tracking disease progression and patient stratification. The need to develop personalized therapeutic strategies is also crucial according to the phenotypic variability observed in FSHD subjects.

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“…Initial efforts to use nanopore sequencing for FSHD, struggled due to lack of read-depth in 4q derived D4Z4 arrays from genomic analyses (Mitsuhashi et al 2017). Development of strategies to enrich sequences of interest using Cas9-targeting has led to improved read-depth for regions of interest (Gilpatrick et al 2020), including the D4Z4 array associated with FSHD (Hiramuki et al 2022) Nanopore sequencing not only opens the door to the full sequencing of the D4Z4 array, but also allows determination of CpG methylation in the array, a critical aspect of genetic analysis for FSHD.…”

Section: Discussionmentioning

confidence: 99%

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

Butterfield

Dunn

Duvall

et al. 2023

Preprint

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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 due to 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.

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

Cited by 18 publications

References 25 publications

D4Z4 Methylation Levels Combined with a Machine Learning Pipeline Highlight Single CpG Sites as Discriminating Biomarkers for FSHD Patients

D4Z4 Methylation Levels Combined with a Machine Learning Pipeline Highlight Single CpG Sites as Discriminating Biomarkers for FSHD Patients

The FSHD jigsaw: are we placing the tiles in the right position?

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

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