Molecular Genetics and Genomics

, Volume 289, Issue 5, pp 1013–1021 | Cite as

Whole dystrophin gene analysis by next-generation sequencing: a comprehensive genetic diagnosis of Duchenne and Becker muscular dystrophy

  • Yan Wang
  • Yao Yang
  • Jing Liu
  • Xiao-Chun Chen
  • Xin Liu
  • Chun-Zhi Wang
  • Xi-Yu He
Methodology article
First Online:
Received:
Accepted:

Abstract

Duchenne/Becker muscular dystrophies are the most frequent inherited neuromuscular diseases caused by mutations of the dystrophin gene. However, approximately 30 % of patients with the disease do not receive a molecular diagnosis because of the complex mutational spectrum and the large size of the gene. The introduction and use of next-generation sequencing have advanced clinical genetic research and might be a suitable method for the detection of various types of mutations in the dystrophin gene. To identify the mutational spectrum using a single platform, whole dystrophin gene sequencing was performed using next-generation sequencing. The entire dystrophin gene, including all exons, introns and promoter regions, was target enriched using a DMD whole gene enrichment kit. The enrichment libraries were sequenced on an Illumina HiSeq 2000 sequencer using paired read 100 bp sequencing. We studied 26 patients: 21 had known large deletion/duplications and 5 did not have detectable large deletion/duplications by multiplex ligation-dependent probe amplification technology (MLPA). We applied whole dystrophin gene analysis by next-generation sequencing to the five patients who did not have detectable large deletion/duplications and to five randomly chosen patients from the 21 who did have large deletion/duplications. The sequencing data covered almost 100 % of the exonic region of the dystrophin gene by ≥10 reads with a mean read depth of 147. Five small mutations were identified in the first five patients, of which four variants were unreported in the dmd.nl database. The deleted or duplicated exons and the breakpoints in the five large deletion/duplication patients were precisely identified. Whole dystrophin gene sequencing by next-generation sequencing may be a useful tool for the genetic diagnosis of Duchenne and Becker muscular dystrophies.

Keywords

Dystrophin gene Next-generation sequencing Genetic diagnosis Duchenne muscular dystrophy 
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Notes

Acknowledgments

We thank all the tested individuals, their families and collaborating clinicians for their participation. This work was supported by the National Natural Science Foundation of China (81300527), the National Science and Technology Support Program of China (2013BA112B00) and Beijing Medical Capital Development Foundation 2012. This study has neither been presented nor submitted or accepted anywhere.

Conflict of interest

The authors declare no conflict of interest.

References

  1. Bennett RR, den Dunnen J, O’Brien KF, Darras BT, Kunkel LM (2001) Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing. BMC Genet 2:17PubMedCrossRefPubMedCentralGoogle Scholar
  2. Bovolenta M, Neri M, Fini S, Fabris M, Trabanelli C, Venturoli A, Martoni E, Bassi E, Spitali P, Brioschi S, Falzarano MS, Rimessi P, Ciccone R, Ashton E, McCauley J, Yau S, Abbs S, Muntoni F, Merlini L, Gualandi F, Ferlini A (2008) A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies. BMC Genom 9:572CrossRefGoogle Scholar
  3. Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 16:11141–11156PubMedCrossRefPubMedCentralGoogle Scholar
  4. Chen WJ, Lin QF, Zhang QJ, He J, Liu XY, Lin MT, Murong SX, Liou CW, Wang N (2013) Molecular analysis of the dystrophin gene in 407 Chinese patients with Duchenne/Becker muscular dystrophy by the combination of multiplex ligation-dependent probe amplification and Sanger sequencing. Clin Chim Acta 423C:35–38CrossRefGoogle Scholar
  5. Deburgrave N, Daoud F, Llense S, Barbot JC, Recan D, Peccate C, Burghes AH, Beroud C, Garcia L, Kaplan JC, Chelly J, Leturcq F (2007) Protein- and mRNA-based phenotype–genotype correlations in DMD/BMD with point mutations and molecular basis for BMD with nonsense and frameshift mutations in the DMD gene. Hum Mutat 28:183–195PubMedCrossRefGoogle Scholar
  6. Dent KM, Dunn DM, von Niederhausern AC, Aoyagi AT, Kerr L, Bromberg MB, Hart KJ, Tuohy T, White S, den Dunnen JT, Weiss RB, Flanigan KM (2005) Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort. Am J Med Genet A 134:295–298PubMedCrossRefGoogle Scholar
  7. Dolinsky LC, de Moura-Neto RS, Falcao-Conceicao DN (2002) DGGE analysis as a tool to identify point mutations, de novo mutations and carriers of the dystrophin gene. Neuromuscul Disord 12:845–848PubMedCrossRefGoogle Scholar
  8. Dwi Pramono ZA, Takeshima Y, Surono A, Ishida T, Matsuo M (2000) A novel cryptic exon in intron 2 of the human dystrophin gene evolved from an intron by acquiring consensus sequences for splicing at different stages of anthropoid evolution. Biochem Biophys Res Commun 267:321–328PubMedCrossRefGoogle Scholar
  9. Emery AE (2002) The muscular dystrophies. Lancet 359:687–695PubMedCrossRefGoogle Scholar
  10. Flanigan KM, von Niederhausern A, Dunn DM, Alder J, Mendell JR, Weiss RB (2003) Rapid direct sequence analysis of the dystrophin gene. Am J Hum Genet 72:931–939PubMedCrossRefPubMedCentralGoogle Scholar
  11. Flanigan KM, Dunn DM, von Niederhausern A, Soltanzadeh P, Gappmaier E, Howard MT, Sampson JB, Mendell JR, Wall C, King WM, Pestronk A, Florence JM, Connolly AM, Mathews KD, Stephan CM, Laubenthal KS, Wong BL, Morehart PJ, Meyer A, Finkel RS, Bonnemann CG, Medne L, Day JW, Dalton JC, Margolis MK, Hinton VJ, Weiss RB (2009) Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat 30:1657–1666PubMedCrossRefPubMedCentralGoogle Scholar
  12. Gatta V, Scarciolla O, Gaspari AR, Palka C, De Angelis MV, Di Muzio A, Guanciali-Franchi P, Calabrese G, Uncini A, Stuppia L (2005) Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA). Hum Genet 117:92–98PubMedCrossRefGoogle Scholar
  13. Grimm T, Kress W, Meng G, Muller CR (2012) Risk assessment and genetic counseling in families with Duchenne muscular dystrophy. Acta Myol 31:179–183PubMedPubMedCentralGoogle Scholar
  14. Hamed SA, Hoffman EP (2006) Automated sequence screening of the entire dystrophin cDNA in Duchenne dystrophy: point mutation detection. Am J Med Genet B Neuropsychiatr Genet 141B:44–50PubMedCrossRefGoogle Scholar
  15. Helderman-van den Enden AT, Straathof CS, Aartsma-Rus A, den Dunnen JT, Verbist BM, Bakker E, Verschuuren JJ, Ginjaar HB (2010) Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients. Neuromuscul Disord 20:251–254PubMedCrossRefGoogle Scholar
  16. Hofstra RM, Mulder IM, Vossen R, de Koning-Gans PA, Kraak M, Ginjaar IB, van der Hout AH, Bakker E, Buys CH, van Ommen GJ, van Essen AJ, den Dunnen JT (2004) DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients. Hum Mutat 23:57–66PubMedCrossRefGoogle Scholar
  17. Lalic T, Vossen RH, Coffa J, Schouten JP, Guc-Scekic M, Radivojevic D, Djurisic M, Breuning MH, White SJ, den Dunnen JT (2005) Deletion and duplication screening in the DMD gene using MLPA. Eur J Hum Genet 13:1231–1234PubMedCrossRefGoogle Scholar
  18. Lenk U, Oexle K, Voit T, Ancker U, Hellner KA, Speer A, Hubner C (1996) A cysteine 3340 substitution in the dystroglycan-binding domain of dystrophin associated with Duchenne muscular dystrophy, mental retardation and absence of the ERG b-wave. Hum Mol Genet 5:973–975PubMedCrossRefGoogle Scholar
  19. Lim BC, Lee S, Shin JY, Kim JI, Hwang H, Kim KJ, Hwang YS, Seo JS, Chae JH (2011) Genetic diagnosis of Duchenne and Becker muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform. J Med Genet 48:731–736PubMedCrossRefGoogle Scholar
  20. Madania A, Zarzour H, Jarjour RA, Ghoury I (2010) Combination of conventional multiplex PCR and quantitative real-time PCR detects large rearrangements in the dystrophin gene in 59% of Syrian DMD/BMD patients. Clin Biochem 43:836–842PubMedCrossRefGoogle Scholar
  21. Marquis-Nicholson R, Lai D, Lan CC, Love JM, Love DR (2013) A streamlined protocol for molecular testing of the DMD Gene within a Diagnostic Laboratory: a combination of array comparative genomic hybridization and bidirectional sequence analysis. ISRN Neurol 2013:908317PubMedCrossRefPubMedCentralGoogle Scholar
  22. Mendell JR, Buzin CH, Feng J, Yan J, Serrano C, Sangani DS, Wall C, Prior TW, Sommer SS (2001) Diagnosis of Duchenne dystrophy by enhanced detection of small mutations. Neurology 57:645–650PubMedCrossRefGoogle Scholar
  23. Mulle JG, Patel VC, Warren ST, Hegde MR, Cutler DJ, Zwick ME (2010) Empirical evaluation of oligonucleotide probe selection for DNA microarrays. PLoS ONE 5:e9921PubMedCrossRefPubMedCentralGoogle Scholar
  24. Murugan S, Chandramohan A, Lakshmi BR (2010) Use of multiplex ligation-dependent probe amplification (MLPA) for Duchenne muscular dystrophy (DMD) gene mutation analysis. Indian J Med Res 132:303–311PubMedGoogle Scholar
  25. Oudet C, Hanauer A, Clemens P, Caskey T, Mandel JL (1992) Two hot spots of recombination in the DMD gene correlate with the deletion prone regions. Hum Mol Genet 1:599–603PubMedCrossRefGoogle Scholar
  26. Roberts RG, Bentley DR, Bobrow M (1993) Infidelity in the structure of ectopic transcripts: a novel exon in lymphocyte dystrophin transcripts. Hum Mutat 2:293–299PubMedCrossRefGoogle Scholar
  27. Sansovic I, Barisic I, Dumic K (2013) Improved detection of deletions and duplications in the DMD gene using the multiplex ligation-dependent probe amplification (MLPA) method. Biochem Genet 51:189–201PubMedCrossRefGoogle Scholar
  28. Shyr D, Liu Q (2013) Next generation sequencing in cancer research and clinical application. Biol Proced Online 15:4PubMedCrossRefPubMedCentralGoogle Scholar
  29. Suminaga R, Takeshima Y, Adachi K, Yagi M, Nakamura H, Matsuo M (2002) A novel cryptic exon in intron 3 of the dystrophin gene was incorporated into dystrophin mRNA with a single nucleotide deletion in exon 5. J Hum Genet 47:196–201PubMedCrossRefGoogle Scholar
  30. Sun H, Chasin LA (2000) Multiple splicing defects in an intronic false exon. Mol Cell Biol 20:6414–6425PubMedCrossRefPubMedCentralGoogle Scholar
  31. Sura T, Eu-ahsunthornwattana J, Pingsuthiwong S, Busabaratana M (2008) Sensitivity and frequencies of dystrophin gene mutations in Thai DMD/BMD patients as detected by multiplex PCR. Dis Markers 25:115–121PubMedCrossRefPubMedCentralGoogle Scholar
  32. Surono A, Takeshima Y, Wibawa T, Ikezawa M, Nonaka I, Matsuo M (1999) Circular dystrophin RNAs consisting of exons that were skipped by alternative splicing. Hum Mol Genet 8:493–500PubMedCrossRefGoogle Scholar
  33. Takeshima Y, Yagi M, Okizuka Y, Awano H, Zhang Z, Yamauchi Y, Nishio H, Matsuo M (2010) Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center. J Hum Genet 55:379–388PubMedCrossRefGoogle Scholar
  34. Todorova A, Todorov T, Georgieva B, Lukova M, Guergueltcheva V, Kremensky I, Mitev V (2008) MLPA analysis/complete sequencing of the DMD gene in a group of Bulgarian Duchenne/Becker muscular dystrophy patients. Neuromuscul Disord 18:667–670PubMedCrossRefGoogle Scholar
  35. Tran VK, Zhang Z, Yagi M, Nishiyama A, Habara Y, Takeshima Y, Matsuo M (2005) A novel cryptic exon identified in the 3′ region of intron 2 of the human dystrophin gene. J Hum Genet 50:425–433PubMedCrossRefGoogle Scholar
  36. Traverso M, Malnati M, Minetti C, Regis S, Tedeschi S, Pedemonte M, Bruno C, Biassoni R, Zara F (2006) Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene. Biochem Biophys Res Commun 339:145–150PubMedCrossRefGoogle Scholar
  37. Trimarco A, Torella A, Piluso G, Maria Ventriglia V, Politano L, Nigro V (2008) Log-PCR: a new tool for immediate and cost-effective diagnosis of up to 85 % of dystrophin gene mutations. Clin Chem 54:973–981PubMedCrossRefGoogle Scholar
  38. White S, Kalf M, Liu Q, Villerius M, Engelsma D, Kriek M, Vollebregt E, Bakker B, van Ommen GJ, Breuning MH, den Dunnen JT (2002) Comprehensive detection of genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable probe hybridization. Am J Hum Genet 71:365–374PubMedCrossRefPubMedCentralGoogle Scholar
  39. White SJ, Aartsma-Rus A, Flanigan KM, Weiss RB, Kneppers AL, Lalic T, Janson AA, Ginjaar HB, Breuning MH, den Dunnen JT (2006) Duplications in the DMD gene. Hum Mutat 27:938–945PubMedCrossRefGoogle Scholar
  40. Xie S, Lan Z, Qu N, Wei X, Yu P, Zhu Q, Yang G, Wang J, Shi Q, Wang W, Yang L, Yi X (2012) Detection of truncated dystrophin lacking the C-terminal domain in a Chinese pedigree by next-generation sequencing. Gene 499:139–142PubMedCrossRefGoogle Scholar
  41. Yan J, Feng J, Buzin CH, Scaringe W, Liu Q, Mendell JR, den Dunnen J, Sommer SS (2004) Three-tiered noninvasive diagnosis in 96% of patients with Duchenne muscular dystrophy (DMD). Hum Mutat 23:203–204PubMedCrossRefGoogle Scholar
  42. Yang J, Li SY, Li YQ, Cao JQ, Feng SW, Wang YY, Zhan YX, Yu CS, Chen F, Li J, Sun XF, Zhang C (2013) MLPA-based genotype–phenotype analysis in 1053 Chinese patients with DMD/BMD. BMC Med Genet 14:29PubMedCrossRefPubMedCentralGoogle Scholar
  43. Zeng F, Ren ZR, Huang SZ, Kalf M, Mommersteeg M, Smit M, White S, Jin CL, Xu M, Zhou DW, Yan JB, Chen MJ, van Beuningen R, den Dunnen J, Zeng YT, Wu Y (2008) Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients. Hum Mutat 29:190–197PubMedCrossRefGoogle Scholar
  44. Zhang Z, Habara Y, Nishiyama A, Oyazato Y, Yagi M, Takeshima Y, Matsuo M (2007) Identification of seven novel cryptic exons embedded in the dystrophin gene and characterization of 14 cryptic dystrophin exons. J Hum Genet 52:607–617PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yan Wang
    • 1
  • Yao Yang
    • 1
  • Jing Liu
    • 2
  • Xiao-Chun Chen
    • 1
  • Xin Liu
    • 1
  • Chun-Zhi Wang
    • 1
  • Xi-Yu He
    • 1
  1. 1.Department of Clinical GeneticsBaYi Children’s Hospital of the General Military Hospital of Beijing PLABeijingPeople’ Republic of China
  2. 2.The Pharmacology DepartmentGeneral Military Hospital of Beijing PLABeijingPeople’ Republic of China

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