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Single-molecule optical mapping enables quantitative measurement of D4Z4 repeats in facioscapulohumeral muscular dystrophy (FSHD)
  1. Yi Dai1,
  2. Pidong Li2,
  3. Zhiqiang Wang3,
  4. Fan Liang2,
  5. Fan Yang2,
  6. Li Fang4,5,
  7. Yu Huang6,
  8. Shangzhi Huang7,
  9. Jiapeng Zhou2,
  10. Depeng Wang2,
  11. Liying Cui1,8,
  12. Kai Wang4,5
  1. 1 Department of Neurology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China
  2. 2 GrandOmics Biosciences, Beijing, China
  3. 3 Department of Neurology and Institute of Neurology, First Affiliated Hospital, Center of Neuroscience, Fujian Medical University, Fuzhou, China
  4. 4 Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
  5. 5 Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA
  6. 6 Department of Medical Genetics, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
  7. 7 Department of Medical Genetics, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China
  8. 8 Neuroscience Center, Chinese Academy of Medical Sciences, Beijing, China, Beijing, China
  1. Correspondence to Professor Kai Wang, Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; wangk{at}


Purpose Facioscapulohumeral 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.

Methods We 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.

Results We 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.

Conclusion While 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.

  • single-molecule optical mapping
  • facioscapulohumeral muscular dystrophy
  • D4Z4
  • FSHD
  • macrosatellite

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  • LC and KW are joint senior authors.

  • YD and PL are joint first authors.

  • Contributors YD, LC and KW planned the study. YD, FY, ZW provided clinical diagnosis of the FSHD patients and performed sample selection, preparation, southern blotting and optical mapping. PL, FL, LF developed the computational pipeline and analysed the single-molecule optical mapping data. YH, SH, JZ, DW advised on the execution of the study and data interpretation. LC and KW are responsible for the overall content and are corresponding authors.

  • Funding This study is in part supported by the CAMS Innovation Fund for Medical Sciences (CIFMS) project number 2016-I2M-1-002.

  • Competing interests PL, FL, FY, JZ and DW are employees and KW was previously a consultant of Grandomics Biosciences.

  • Patient consent for publication Obtained.

  • Ethics approval The study was approved by the Institutional Review Board of the Peking Union Medical College of the Chinese Academy of Medical Sciences.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available in a public, open-access repository. Data are available on reasonable request and institutional data use agreement.

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