Detection of Duchene muscular dystrophy carriers with quantitative fluorescent polymerase chain reaction
Detection of Duchene muscular dystrophy carriers with quantitative fluorescent polymerase chain reaction
Aim: Duchenne Muscular Dystrophy (DMD) is an X-linked, progressive, lethal neuromuscular disorder affecting 1/3500 live-bornmales. Mutations occur in the dystrophin gene, which is located at Xp21.2. Partial gene deletions occur in two “hot-spot” regions, andcan be responsible for up to 60-65% DMD cases, while 5-10% of the cases are caused from clustered gene duplications. Mutationscan be inherited from female carriers (2/3) or be de-novo mutations (1/3). Deletions can be easily detected in affected males viamultiplex PCR or MLPA. On the contrary, determining the status of female carriers is difficult. The aim of this study is to optimize thegene-dosage method using quantitative fluorescent PCR.Material and Methods: Fluorescently labeled primers are used for amplification and automated detection of amplicons and aredesigned in multiplex format. The primers contain eighteen exons located within “hot-spot” regions. A promoter region and STRmarkers are also included in the test as internal controls and for linkage analysis. This is followed by a PCR automated geneticanalyzer for the detection of PCR products. This study includes twenty-four families, each with a previously diagnosed member.Results: Results showed the same correlation as was previously reported in nineteen patients, whereas three patients had an extraexon deletion and one patient had one less exon deletion than previously reported. In nineteen families, the mothers were carriers,and in five families, the mothers were not carriers.Conclusion: As a conclusion for carrier screening in DMD patients, quantitative fluorescent PCR is a fast, reproducible and robustmethod can be used for detection of deletions.
___
- 1. Alcantara MA, Garcia-Cavazos R, Hernandez UE, et al.
Carrier detection and prenatal molecular diagnosis in a
Duchenne muscular dystrophy family without any affected
relative available. Ann Genet 2001;44:149-53.
- 2. Birnkrant DJ, Bushby K, Bann CM, et al. Diagnosis and
management of Duchenne muscular dystrophy, part 1:
diagnosis, and neuromuscular, rehabilitation, endocrine,
and gastrointestinal and nutritional management. Lancet
Neurol 2018;17:251-67.
- 3. Frisso G, Carsana A, Tinto N, et al. Direct detection of exon
deletions/duplications in female carriers of and male
patients with Duchenne/Becker muscular dystrophy. Clin
Chem 2004;50:1435-8.
- 4. Hung CC, Chen CP, Lin SP, et al. Quantitative assay of
deletion or duplication genotype by capillary electrophoresis
system: Application in Prader-Willi syndrome and Duchenne
muscular dystrophy. Clin Chem 2006;52:2203-10.
- 5. Grimm T, Muller B, Dreier M, et al. Hot spot of recombination
within DXS164 in the Duchenne muscular dystrophy gene.
Am J Hum Genet 1989;45:368-72.
- 6. Echigoya Y, Lim KRQ, Nakamura A, et al. Multiple exon
skipping in the duchenne muscular dystrophy hot spots:
Prospects and Challenges. J Pers Med 2018;8.
- 7. Gatta V, Scarciolla O, Gaspari AR, et al. Identification of
deletions and duplications of the DMD gene in affected
males and carrier females by multiple ligation probe
amplification (MLPA). Hum Genet 2005;117:92-8.
- 8. Aartsma-Rus A, Ginjaar IB, Bushby K. The importance of
genetic diagnosis for Duchenne muscular dystrophy. J Med
Genet 2016;53:145-51.
- 9. Nakamura A. Mutation-Based Therapeutic Strategies for
Duchenne Muscular Dystrophy: From Genetic Diagnosis to
Therapy. J Pers Med 2019;9.
- 10. Falzarano MS, Scotton C, Passarelli C, et al. Duchenne
Muscular Dystrophy: From Diagnosis to Therapy. Molecules
2015;20:18168-84.
- 11. Chamberlain JS, Gibbs RA, Ranier JE, et al. Deletion screening
of the Duchenne muscular dystrophy locus via multiplex
DNA amplification. Nucleic Acids Res 1988;16:11141-56.
- 12. Henegariu O, Heerema NA, Dlouhy SR, et al. Multiplex PCR:
critical parameters and step-by-step protocol. Biotechniques
1997;23:504-11.
- 13. Dolinsky LC, de Moura-Neto RS, Falcao-Conceicao DN.
DGGE analysis as a tool to identify point mutations, de novo
mutations and carriers of the dystrophin gene. Neuromuscul
Disord 2002;12:845-8.
- 14. Ligon AH, Kashork CD, Richards CS, et al. Identification
of female carriers for Duchenne and Becker muscular
dystrophies using a FISH-based approach. Eur J Hum
Genet.2000;8:293-8.
- 15. Chaturvedi LS, Srivastava S, Mukherjee M, et al. Carrier
detection in non-deletional Duchenne/Becker muscular
dystrophy families using polymorphic dinucleotide (CA)
repeat loci of dystrophin gene. Indian J Med Res 2001;113:19-
25.
- 16. Janssen B, Hartmann C, Scholz V, et al. MLPA analysis
for the detection of deletions, duplications and complex
rearrangements in the dystrophin gene: potential and pitfalls.
Neurogenetics 2005;6:29-35.
- 17. Lai KK, Lo IF, Tong TM, et al. Detecting exon deletions and
duplications of the DMD gene using Multiplex Ligationdependent
Probe Amplification (MLPA). Clin Biochem.
2006;39:367-72.
- 18. Elnifro EM, Ashshi AM, Cooper RJ, et al. Multiplex PCR:
optimization and application in diagnostic virology. Clin
Microbiol Rev 2000;13:559-70.