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Open Access Highly Accessed Research article

Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing

Richard R Bennett1*, Johan den Dunnen2, Kristine F O'Brien3, Basil T Darras4 and Louis M Kunkel135

Author affiliations

1 Division of Genetics, Children's Hospital, Boston, Massachusetts, USA

2 Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, Nederland

3 Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

4 Department of Neurology, Children's Hospital, Boston, Massachusetts, USA

5 Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA

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Citation and License

BMC Genetics 2001, 2:17  doi:10.1186/1471-2156-2-17

Published: 17 October 2001

Abstract

Background

Currently molecular diagnostic laboratories focus only on the identification of large deletion and duplication mutations (spanning one exon or more) for Duchenne Muscular Dystrophy (DMD) yielding 65% of causative mutations. These mutations are detected by an existing set of multiplexed polymerase chain reaction (PCR) primer pairs. Due to the large size of the dystrophin gene (79 exons), finding point mutations (substitutions, deletions or insertions of one or several nucleotides) has been prohibitively expensive and laborious. The aim of this project was to develop an effective and convenient method of finding all, or most, mutations in the dystrophin gene with only a moderate increase in cost.

Results

Using denaturing high performance liquid chromatography (DHPLC) screening and direct sequencing, 86 PCR amplicons of genomic DNA from the dystrophin gene were screened for mutations in eight patients diagnosed with DMD who had tested negative for large DNA rearragements. Mutations likely to be disease-causative were found in six of the eight patients. All 86 amplicons from the two patients in whom no likely disease-causative mutations were found were completely sequenced and only polymorphisms were found.

Conclusions

We have shown that it is now feasible for clinical laboratories to begin testing for both point mutations and large deletions/duplications in the dystrophin gene. The detection rate will rise from 65% to greater than 92% with only a moderate increase in cost.