title: 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.
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.
authors.affiliation: 1 Division of Translational and Systems Medicine, Warwick Medical School, UK.
authors.forename: Anne
authors.initials: A
authors.lastname: Reiman
authors.affiliation: 2 Pathology Service, University Hospital Coventry and Warwickshire, UK.
authors.forename: Sarojini
authors.initials: S
authors.lastname: Pandey
authors.affiliation: 3 Systems Biology, University of Warwick, UK.
authors.forename: Kate L
authors.initials: KL
authors.lastname: Lloyd
authors.affiliation: 3 Systems Biology, University of Warwick, UK.
authors.forename: Nigel
authors.initials: N
authors.lastname: Dyer
authors.affiliation: 4 Department of Endocrinology and Diabetes, University Hospital Coventry and Warwickshire, UK.
authors.forename: Mike
authors.initials: M
authors.lastname: Khan
authors.affiliation: 5 Molecular Diagnostics Group, Randox Laboratories Limited, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Crockard
authors.affiliation: 5 Molecular Diagnostics Group, Randox Laboratories Limited, UK.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Latten
authors.affiliation: 5 Molecular Diagnostics Group, Randox Laboratories Limited, UK.
authors.forename: Tracey L
authors.initials: TL
authors.lastname: Watson
authors.affiliation: 2 Pathology Service, University Hospital Coventry and Warwickshire, UK.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Cree
authors.affiliation: 1 Division of Translational and Systems Medicine, Warwick Medical School, UK.|2 Pathology Service, University Hospital Coventry and Warwickshire, UK.
authors.forename: Dimitris K
authors.initials: DK
authors.lastname: Grammatopoulos
mesh_terms: D000328:Adult; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D015331:Cohort Studies; D004252:DNA Mutational Analysis; D005260:Female; D015870:Gene Expression; D005820:Genetic Testing; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D000071449:Proprotein Convertase 9 / Q000235:genetics*; D011973:Receptors, LDL / Q000235:genetics*; D006113:United Kingdom
chemical_list: D001055:Apolipoproteins B; C573412:LDLR protein, human; D011973:Receptors, LDL; D000071449:Proprotein Convertase 9
keywords: Next-generation sequencing; apolipoprotein B; familial hypercholesterolaemia mutation; low-density lipoprotein receptor; molecular screening