© 2005 BMJ Publishing Group Ltd & Association of Clinical Pathologists
ORIGINAL ARTICLE
Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease
1 Department of Molecular and Clinical Genetics, Royal Prince Alfred Hospital and Central Clinical School, The University of Sydney, Missenden Road, Camperdown, NSW 2050, Australia
2 Department of Cardiology, Royal Prince Alfred Hospital and Central Clinical School
3 School of Medical Sciences, University of Sydney, Camperdown, NSW 2006, Australia
Correspondence to:
Correspondence to:
Dr B Yu
Department of Molecular and Clinical Genetics, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia; bingy{at}med.usyd.edu.au
Aims: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND).
Methods: The coding sequence and intronexon boundaries of the cardiac ß myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods.
Results: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening.
Conclusions: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.
Abbreviations: DHPLC, denaturing high performance liquid chromatography; FHC, familial hypertrophic cardiomyopathy; MND, motor neurone disease; MYH7, cardiac ß-myosin heavy chain gene; PCR, polymerase chain reaction; PVR, poliovirus receptor gene; SNP, single nucleotide polymorphism
Keywords: DNA variant analysis; denaturing high performance liquid chromatography; familial hypertrophic cardiomyopathy; motor neurone disease; high throughput
![]()
CiteULike
Complore
Connotea
Del.icio.us
Digg
Reddit
Technorati What's this?
This article has been cited by other articles:
-
Huang, X. X., Bernerd, F., Halliday, G. M.
(2009). Ultraviolet A within Sunlight Induces Mutations in the Epidermal Basal Layer of Engineered Human Skin. Am. J. Pathol.
174: 1534-1543
[Abstract] [Full Text] -
Olivotto, I., Girolami, F., Ackerman, M. J., Nistri, S., Bos, J. M., Zachara, E., Ommen, S. R., Theis, J. L., Vaubel, R. A., Re, F., Armentano, C., Poggesi, C., Torricelli, F., Cecchi, F.
(2008). Myofilament Protein Gene Mutation Screening and Outcome of Patients With Hypertrophic Cardiomyopathy. Mayo Clin Proc.
83: 630-638
[Abstract] [Full Text] -
Waldmuller, S., Muller, M., Rackebrandt, K., Binner, P., Poths, S., Bonin, M., Scheffold, T.
(2008). Array-Based Resequencing Assay for Mutations Causing Hypertrophic Cardiomyopathy. Clin. Chem.
54: 682-687
[Abstract] [Full Text] -
Levano, S., Keller, D., Schobinger, E., Urwyler, A., Girard, T.
(2008). Rapid and Accurate Detection of Atypical and Kalow Variants in the Butyrylcholinesterase Gene Using Denaturing High Performance Liquid Chromatography. Anesth. Analg.
106: 147-151
[Abstract] [Full Text]
Register for free content
The full back archive is now available for all BMJ Journals. Institutional subscribers may access the entire archive as part of their subscription. Personal subscribers will also have access to all content when logged in. Non-subscribers who register have free access to all articles published before 2006 right back to volume 1 issue 1. Register here to access the free archive of all BMJ Journals.
Don't forget to sign up for content alerts so you keep up to date with all the articles as they are published.
