Journal of Genetic Medicine

Korean Society of Medical Genetics and Genomics (대한의학유전학회)
권호 표지
DOI QR코드

DOI QR Code

Southern Analysis after Long-range PCR: Clinical Application in Korean Patients with Myotonic Dystrophy 1

  • Yum, Mi-Sun (Department of Pediatrics, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine) ;
  • Lee, Beom Hee (Department of Pediatrics, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine) ;
  • Kim, Gu-Hwan (Medical Genetics Center, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine) ;
  • Lee, Jin-Joo (Medical Genetics Center, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine) ;
  • Choi, Seung Hoon (Medical Genetics Center, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine) ;
  • Lee, Joo Yeon (Medical Genetics Center, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine) ;
  • Kim, Jae-Min (Medical Genetics Center, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine) ;
  • Kim, Yoo-Mi (Department of Pediatrics, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine) ;
  • Ko, Tae-Sung (Department of Pediatrics, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine) ;
  • Yoo, Han-Wook (Department of Pediatrics, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine)
  • Received : 2013.05.28
  • Accepted : 2013.06.12
  • Published : 2013.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose: Myotonic dystrophy 1 (DM1, OMIM 160900) is an autosomal-dominant muscular disorder caused by an expansion of CTG repeats in the 3' UTR of the DMPK gene. Variable expansions of CTG repeats preclude the accurate determination of repeat size. We tried to show the clinical and analytical validity of the application of Southern blotting after long-range PCR was demonstrated in Korean DM1 patients. Materials and Methods: The Southern blotting of long-range PCR was applied to 1,231 cases with clinical suspicion of DM1, between 2000 and 2011. PCR was performed using genomic DNA with forward 5'-CAGTTCACAACCGCTCCGAGC-3' and reverse 5'-CGTGGAGGATGGAACACGGAC-3' primers. Subsequently, the PCR fragments were subjected to gel electrophoresis, capillary transfer to a nylon membrane, hybridization with a labeled (CAG)10 probe. The correlation between clinical manifestations and the CTG repeat expansions were analyzed. Results: Among a total of 1,231 tested cases, 642 individuals were diagnosed with DM1 and the range of the detected expansion was 50 to 2,500 repeats; fourteen cases with mild DM1 ($75{\pm}14$ repeats), 602 cases with classical DM1 ($314{\pm}143$ repeats), and 26 cases with congenital DM1 ($1,219{\pm}402$ repeats). The positive and negative predictive values were 100%. The age at test requested and the CTG repeat numbers were inversely correlated (R=-0.444, P<0.01). Conclusion: This study indicates that Southern blotting after long-range PCR is a reliable diagnostic method DM1.

Keywords

References

  1. Kamsteeg EJ, Kress W, Catalli C, Hertz JM, Witsch-Baumgartner M, Buckley MF, et al. Best practice guidelines and recommendations on the molecular diagnosis of myotonic dystrophy types 1 and 2. Eur J Hum Genet 2012;20:1203-8. https://doi.org/10.1038/ejhg.2012.108
  2. Turner C, Hilton-Jones D. The myotonic dystrophies: diagnosis and management. J Neurol Neurosurg Psychiatry 2010;81:358-67. https://doi.org/10.1136/jnnp.2008.158261
  3. Lavedan C, Hofmann-Radvanyi H, Shelbourne P, Rabes JP, Duros C, Savoy D, et al. Myotonic dystrophy: size- and sex-dependent dynamics of CTG meiotic instability, and somatic mosaicism. Am J Hum Genet 1993;52:875-83.
  4. Harley HG, Rundle SA, MacMillan JC, Myring J, Brook JD, Crow S, et al. Size of the unstable CTG repeat sequence in relation to phenotype and parental transmission in myotonic dystrophy. Am J Hum Genet 1993; 52:1164-74.
  5. Gennarelli M, Novelli G, Andreasi Bassi F, Martorell L, Cornet M, Menegazzo E, et al. Prediction of myotonic dystrophy clinical severity based on the number of intragenic [CTG]n trinucleotide repeats. Am J Med Genet 1996;65:342-7. https://doi.org/10.1002/(SICI)1096-8628(19961111)65:4<342::AID-AJMG18>3.0.CO;2-U
  6. Radvansky J, Kadasi L. The expanding world of myotonic dystrophies: how can they be detected? Genet Test Mol Biomarkers 2010;14:733-41. https://doi.org/10.1089/gtmb.2010.0073
  7. Falk M, Vojtísková M, Lukás Z, Kroupová I, Froster U. Simple procedure for automatic detection of unstable alleles in the myotonic dystrophy and Huntington's disease loci. Genet Test 2006;10:85-97. https://doi.org/10.1089/gte.2006.10.85
  8. New nomenclature and DNA testing guidelines for myotonic dystrophy type 1 (DM1). The International Myotonic Dystrophy Consortium (IDMC). Neurology 2000;54:1218-21. https://doi.org/10.1212/WNL.54.6.1218
  9. Harper PS. Myotonic dystrophy. 3rd ed. London: W.B.Sounders, 2001.
  10. Zühlke C, Atici J, Martorell L, Gembruch U, Kohl M, Göpel W, et al. Rapid detection of expansions by PCR and non-radioactive hybridization: application for prenatal diagnosis of myotonic dystrophy. Prenat Diagn 2000;20:66-9. https://doi.org/10.1002/(SICI)1097-0223(200001)20:1<66::AID-PD745>3.0.CO;2-H
  11. Skrzypczak-Zielinska M, Sulek-Piatkowska A, Mierzejewski M, Froster UG. New analysis method of myotonic dystrophy 1 based on quantitative fluorescent polymerase chain reaction. Genet Test Mol Biomarkers 2009;13:651-5. https://doi.org/10.1089/gtmb.2009.0050
  12. Cheng S, Barceló JM, Korneluk RG. Characterization of large CTG repeat expansions in myotonic dystrophy alleles using PCR. Hum Mutat 1996; 7:304-10. https://doi.org/10.1002/(SICI)1098-1004(1996)7:4<304::AID-HUMU3>3.0.CO;2-8
  13. Bird TD. Myotonic Dystrophy Type 1. http://www.ncbi.nlm.nih.gov/ books/NBK1165/
  14. Pearson CE, Sinden RR. Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci. Biochemistry 1996;35:5041-53. https://doi.org/10.1021/bi9601013
  15. Radvansky J, Ficek A, Kadasi L. Upgrading molecular diagnostics of myotonic dystrophies: multiplexing for simultaneous characterization of the DMPK and ZNF9 repeat motifs. Mol Cell Probes 2011;25:182-5. https://doi.org/10.1016/j.mcp.2011.04.006
  16. Hamzi K, Bellayou H, Slassi I, Nadifi S. A rapid polymerase chain reactionbased test for screening Steinert's disease (DM1). Neurol India 2010;58: 99-102. https://doi.org/10.4103/0028-3886.60411
  17. Kakourou G, Dhanjal S, Mamas T, Serhal P, Delhanty JD, SenGupta SB. Modification of the triplet repeat primed polymerase chain reaction method for detection of the CTG repeat expansion in myotonic dystrophy type 1: application in preimplantation genetic diagnosis. Fertil Steril 2010;94:1674-9. https://doi.org/10.1016/j.fertnstert.2009.10.050
  18. Day JW, Ricker K, Jacobsen JF, Rasmussen LJ, Dick KA, Kress W, et al. Myotonic dystrophy type 2: molecular, diagnostic and clinical spectrum. Neurology 2003;60:657-64. https://doi.org/10.1212/01.WNL.0000054481.84978.F9
  19. Mahadevan M, Tsilfidis C, Sabourin L, Shutler G, Amemiya C, Jansen G, et al. Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science 1992;255:1253-5. https://doi.org/10.1126/science.1546325