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
| Autor: | Mike Khan, Tracey L Watson, Ian A Cree, Dimitris K. Grammatopoulos, Sarojini Pandey, Martin Crockard, Mark J Latten, Nigel P. Dyer, Kate L Lloyd, Anne Reiman |
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| Rok vydání: | 2016 |
| Předmět: |
Adult
Male 0301 basic medicine Apolipoprotein B Concordance DNA Mutational Analysis Clinical Biochemistry Gene Expression Biology medicine.disease_cause DNA sequencing Cohort Studies Hyperlipoproteinemia Type II 03 medical and health sciences Multiplex polymerase chain reaction medicine Humans Genetic Testing Child Apolipoproteins B Oligonucleotide Array Sequence Analysis Genetic testing Genetics Mutation Base Sequence medicine.diagnostic_test High-Throughput Nucleotide Sequencing General Medicine Ion semiconductor sequencing Middle Aged Proprotein convertase United Kingdom 030104 developmental biology Receptors LDL Child Preschool biology.protein Female Proprotein Convertase 9 Multiplex Polymerase Chain Reaction |
| Zdroj: | Annals of Clinical Biochemistry: International Journal of Laboratory Medicine. 53:654-662 |
| ISSN: | 1758-1001 0004-5632 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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