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Use of denaturing HPLC to provide efficient detection of mutations causing familial hypercholesterolemia
Olaf A Bodamer1, Dan Bercovich, Michael Schlabach
1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. olaf.bodamer@univie.ac.at
Insights
Denaturing HPLC (DHPLC) efficiently detects mutations causing familial hypercholesterolemia (FH). This method, combined with DNA sequencing, offers a sensitive approach for diagnosing this common genetic disorder.
Area of Science:
- Genetics
- Molecular Biology
- Cardiovascular Disease
Background:
- Familial hypercholesterolemia (FH) is a common genetic disorder caused by LDL receptor (LDLR) gene mutations.
- It leads to significant cardiovascular morbidity and mortality.
- Current diagnostic methods are imprecise, labor-intensive, and expensive.
Purpose of the Study:
- To evaluate denaturing HPLC (DHPLC) as a sensitive and efficient method for detecting LDLR gene mutations in FH.
- To improve the molecular diagnosis of FH.
Main Methods:
- PCR amplification of all LDLR exons, intron boundaries, and promoter region.
- Mutation screening using DHPLC followed by direct sequencing of aberrant fragments.
- Analysis of nine FH individuals, two FH cell lines, and 50 controls.
Main Results:
- Previously reported disease-causing LDLR mutations were identified in 8 of 9 FH individuals and both cell lines.
- No mutations were found in control individuals.
- One FH individual had an unreported 5'-untranslated region change; several polymorphisms were identified.
Conclusions:
- DHPLC is a viable method for detecting FH-causing mutations.
- DHPLC combined with DNA sequencing is a sensitive and efficient diagnostic approach for FH.
- This method can aid in the early diagnosis and management of FH.
Background:
Autosomal dominant familial hypercholesterolemia (FH) attributable to mutations in the LDL receptor (LDLR) gene is one of the most common genetic disorders associated with significant morbidity and mortality. Definitive diagnosis would help to initiate appropriate treatment to prevent premature cardiovascular disease. Currently, clinical diagnosis of FH is imprecise, and molecular diagnosis is labor-intensive and expensive because of the size of the LDLR gene and number of coding exons.
Methods:
We used PCR to amplify all exons, including exon/intron boundaries, and the promoter of the LDLR gene. Nine individuals from five families with typical findings for a clinical diagnosis of heterozygous FH, 2 heterozygous FH cell lines, and 50 control individuals were screened for mutations by denaturing HPLC (DHPLC) followed by direct sequencing of aberrantly migrating fragments.
Results:
Mutations that were previously reported to be disease causing were identified in eight of nine individuals with FH and both cell lines (V502M, C146X, E207X, C660X, C646Y, and delG197), but none were found in controls. The one individual with FH in whom no mutation was found had a previously unreported change in the 5'-untranslated region of unknown significance. In addition, we identified several previously reported polymorphism both in controls and individuals with FH.
Conclusions:
DHPLC can be used to detect mutations causing FH. On the basis of our current experience with DHPLC, this method combined with confirmatory DNA sequencing is likely to be sensitive and efficient.