title: Use of denaturing HPLC to provide efficient detection of mutations causing familial hypercholesterolemia.
abstract: 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.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. olaf.bodamer@univie.ac.at
authors.forename: Olaf A
authors.initials: OA
authors.lastname: Bodamer
authors.forename: Dan
authors.initials: D
authors.lastname: Bercovich
authors.forename: Michael
authors.initials: M
authors.lastname: Schlabach
authors.forename: Christie
authors.initials: C
authors.lastname: Ballantyne
authors.forename: Danièle
authors.initials: D
authors.lastname: Zoch
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Beaudet
mesh_terms: 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
chemical_list: D011973:Receptors, LDL