Use of denaturing HPLC to provide efficient detection of mutations causing familial hypercholesterolemia.
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.
Bodamer
Olaf A
OA
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. olaf.bodamer@univie.ac.at
Bercovich
Dan
D
Schlabach
Michael
M
Ballantyne
Christie
C
Zoch
Danièle
D
Beaudet
Arthur L
AL
D000328:Adult; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis; D005091:Exons; D005260:Female; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011973:Receptors, LDL / Q000235:genetics; D012680:Sensitivity and Specificity
D011973:Receptors, LDL