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Dystrophin gene analysis on 130 patients with Duchenne muscular dystrophy with a special reference to muscle mRNA
M Ikezawa1, N Minami, M Takahashi
1Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan. ikezawa@ncnaxp.ncnp.go.jp
Abstract:
On dystrophin gene analysis by multiplex polymerase chain reaction (PCR), 76 of 130 (58.5%) Japanese patients with Duchenne muscular dystrophy had a deletion or duplication in genomic DNA. Of the remaining 54 patients who had no identifiable gene mutations, muscle biopsy tissue was available in 16 for RNA extraction. The full length of the coding regions of dystrophin cDNA was amplified in 10 fragments by reverse transcription nested PCR (RT-PCR). Five of 16 patients (31%) had dystrophin cDNA of abnormal size. One patient had a deletion, and two duplications that were not covered by multiplex PCR, one an exon-skipping of exon 51 caused by a 5' consensus splice site mutation of intron 51, and one 172 bp or 202 bp insertion in the cDNA between exon 25 and 26. Nested RT-PCR from the total RNA extracted from muscle biopsy was useful for screening patients who had no identifiable gene abnormality by multiplex PCR.
Insights
Multiplex PCR identified gene deletions/duplications in 58.5% of Duchenne muscular dystrophy patients. Further RNA analysis revealed additional mutations, improving diagnostic yield for genetic disorders.
Area of Science:
- Genetics
- Molecular Biology
- Neuromuscular Disorders
Background:
- Duchenne muscular dystrophy (DMD) is a severe genetic disorder.
- Accurate genetic diagnosis is crucial for patient management.
- Multiplex PCR is a common method for detecting dystrophin gene mutations.
Purpose of the Study:
- To evaluate the utility of multiplex PCR and RT-PCR for diagnosing dystrophin gene mutations in Japanese DMD patients.
- To identify mutations missed by initial multiplex PCR screening.
Main Methods:
- Genomic DNA analysis using multiplex polymerase chain reaction (PCR).
- RNA extraction from muscle biopsy tissue for patients with no identifiable DNA mutations.
- Dystrophin cDNA amplification via reverse transcription nested PCR (RT-PCR).
Main Results:
- Multiplex PCR detected deletions or duplications in 58.5% (76/130) of Japanese DMD patients.
- In patients negative by multiplex PCR, RT-PCR identified abnormal dystrophin cDNA in 31% (5/16).
- Identified mutations included deletions, duplications, exon skipping, and cDNA insertions not detected by multiplex PCR.
Conclusions:
- Multiplex PCR is effective for initial DMD mutation screening.
- Nested RT-PCR on muscle biopsy RNA is valuable for detecting mutations missed by genomic analysis.
- Combined approaches enhance the diagnostic accuracy for Duchenne muscular dystrophy.