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Logo of bmcgeneBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Genetics
 
BMC Genet. 2001; 2: 17.
Published online Oct 17, 2001. doi:  10.1186/1471-2156-2-17
PMCID: PMC59832
Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing
Richard R Bennett,corresponding author1 Johan den Dunnen,2 Kristine F O'Brien,3 Basil T Darras,4 and Louis M Kunkel1,3,5
1Division of Genetics, Children's Hospital, Boston, Massachusetts, USA
2Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, Nederland
3Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
4Department of Neurology, Children's Hospital, Boston, Massachusetts, USA
5Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA
corresponding authorCorresponding author.
Richard R Bennett: bennett/at/rascal.med.harvard.edu; Johan den Dunnen: ddunnen/at/lumc.nl; Kristine F O'Brien: kfobrien/at/mac.com; Basil T Darras: darras_b/at/Hub.TCH.Harvard.edu; Louis M Kunkel: kunkel/at/genetics.med.harvard.edu
Received August 22, 2001; Accepted October 17, 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.
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