title: Microfluidic amplification as a tool for massive parallel sequencing of the familial hypercholesterolemia genes.
abstract: BACKGROUND
  Familial hypercholesterolemia (FH) is an autosomal dominant disorder that affects cholesterol metabolism and is an important risk factor for heart disease. Three different genes were causally linked to this disorder: LDLR (low density lipoprotein receptor), APOB [apolipoprotein B (including Ag(x) antigen)], and PCSK9 (proprotein convertase subtilisin/kexin type 9). We evaluated a new amplicon preparation tool for resequencing these genes on next generation sequencing (NGS) platforms.
  
  
  METHODS
  For the 3 genes, 38 primer pairs were designed and loaded on the Fluidigm Access Array, a microfluidic array in which a PCR was performed. We amplified 144 DNA samples (73 positive controls and 71 patient samples) and performed 3 sequencing runs on a GS FLX Titanium system from Roche 454, using pyrosequencing. Data were analyzed with the SeqNext module of the Sequence Pilot software.
  
  
  RESULTS
  From the 38 amplicons, 37 were amplified successfully, without any further optimization. Sequencing resulted in a mean coverage of the individual amplicons of 71-fold, 74-fold, and 117-fold for the 3 runs, respectively. In the positive controls, all known mutations were identified. In 29% of the patient samples, a pathogenic point mutation or small deletion/insertion was found. Large rearrangements were not detectable with NGS, but were picked up by multiplex ligation-dependent probe amplification.
  
  
  CONCLUSIONS
  Combining a microfluidic amplification system with massive parallel sequencing is an effective method for mutation scanning in FH patients, which can be implemented in diagnostics. For data analysis, we propose a minimum variant frequency threshold of 20% and a minimum coverage of 25-fold.
authors.affiliation: Laboratory for Molecular Diagnosis, Center for Human Genetics, University of Leuven, Leuven, Belgium.
authors.forename: Silke
authors.initials: S
authors.lastname: Hollants
authors.forename: Egbert J W
authors.initials: EJ
authors.lastname: Redeker
authors.forename: Gert
authors.initials: G
authors.lastname: Matthijs
mesh_terms: D001055:Apolipoproteins B / Q000235:genetics; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics*; D046210:Microfluidic Analytical Techniques; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000235:genetics; D011973:Receptors, LDL / Q000235:genetics; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000235:genetics
chemical_list: D001055:Apolipoproteins B; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases