Molecular testing for familial hypercholesterolaemia-associated mutations in a UK-based cohort: Development of an NGS-based method and comparison with multiplex polymerase chain reaction and oligonucleotide arrays

Anne Reiman, Sarojini Pandey, Kate L Lloyd, Nigel Dyer, Mike Khan, Martin Crockard, Mark J Latten, Tracey L Watson, Ian A Cree, Dimitris K Grammatopoulos

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Background Detection of disease-associated mutations in patients with familial hypercholesterolaemia is crucial for early interventions to reduce risk of cardiovascular disease. Screening for these mutations represents a methodological challenge since more than 1200 different causal mutations in the low-density lipoprotein receptor has been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories. Methods Using primers targeting, the low-density lipoprotein receptor, apolipoprotein B, and proprotein convertase subtilisin/kexin type 9, we developed a novel Ion Torrent-based targeted re-sequencing method. We validated this in a West Midlands-UK small cohort of 58 patients screened in parallel with other mutation-targeting methods, such as multiplex polymerase chain reaction (Elucigene FH20), oligonucleotide arrays (Randox familial hypercholesterolaemia array) or the Illumina next-generation sequencing platform. Results In this small cohort, the next-generation sequencing method achieved excellent analytical performance characteristics and showed 100% and 89% concordance with the Randox array and the Elucigene FH20 assay. Investigation of the discrepant results identified two cases of mutation misclassification of the Elucigene FH20 multiplex polymerase chain reaction assay. A number of novel mutations not previously reported were also identified by the next-generation sequencing method. Conclusions Ion Torrent-based next-generation sequencing can deliver a suitable alternative for the molecular investigation of familial hypercholesterolaemia patients, especially when comprehensive mutation screening for rare or unknown mutations is required.

Original languageEnglish
Pages (from-to)654-662
Number of pages9
JournalAnnals of Clinical Biochemistry
Volume53
Issue number6
Early online date8 Jan 2016
DOIs
Publication statusPublished - 1 Nov 2016
Externally publishedYes

Keywords

  • Adult
  • Apolipoproteins B
  • Base Sequence
  • Child
  • Child, Preschool
  • Cohort Studies
  • DNA Mutational Analysis
  • Female
  • Gene Expression
  • Genetic Testing
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Hyperlipoproteinemia Type II
  • Male
  • Middle Aged
  • Multiplex Polymerase Chain Reaction
  • Mutation
  • Oligonucleotide Array Sequence Analysis
  • Proprotein Convertase 9
  • Receptors, LDL
  • United Kingdom
  • Comparative Study
  • Journal Article

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