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Diagnostic Strategy for the Detection of Dystrophin Gene Mutations in Asian Patients and Carriers Using Immortalized Cell Lines

Department of Paediatrics Faculty of Medicine National University of Singapore Singapore

Hwee Hoon Khng, BSc Hons

Department of Paediatrics Faculty of Medicine National University of Singapore Singapore

Poh Sim Low, MBBS, MMed, FRCP Ed MD, FRCPCH, FRACP, BSc Hons

Department of Paediatrics Faculty of Medicine National University of Singapore Singapore

Poh San Lai, PdD

Department of Paediatrics Faculty of Medicine National University of Singapore Singapore, paelaips{at}nus.edu.sg

Duchenne muscular dystrophy and Becker muscular dystrophy are X-linked recessive diseases of muscle degeneration caused by mutations in the dystrophin gene. More than half of our local Asian patients have point mutations that cannot be detected by conventional multiplex polymerase chain reaction deletion screening. This study aimed to develop mutational screening and carrier detection for Duchenne and Becker muscular dystrophy using protein truncation analysis from Epstein-Barr virus—transformed lymphocyte cell lines. Messenger ribonucleic acid was extracted from fresh lymphocytes and Epstein-Barr virus—transformed lymphocyte cell lines of 14 patients. Reverse transcriptase polymerase chain reaction was performed in 11 overlapping segments, followed by in vitro protein translation and truncation analysis. DNA sequencing was carried out for the corresponding complementary DNA regions, which showed aberrant truncated protein products. Carrier studies using this method were also performed for two families. Half of the patients had frame-shifting deletions, and the remaining seven patients showed point mutations, of which four were novel. These mutations were detected in messenger ribonucleic acid extracted from both fresh lymphocytes and Epstein-Barr virus—transformed lymphocyte cell lines. Carrier status was confirmed in one family and was found to be negative in the other family studied. Protein truncation analysis is an efficient method of screening truncating point mutations from immortalized lymphocyte cell lines from patients. This approach not only serves to prove the pathogenicity of both deletion- and nondeletion-type mutations; it is also effective for carrier detection. The use of such cell lines obviates the need for repeated blood and muscle sampling in patients and offers a perpetual source of messenger ribonucleic acid that can be used long after the patient's demise. (J Child Neurol 2006;21:150—155; DOI 10.2310/7010.2006.00029).

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