ANNALS EXPRESS: Molecular testing for familial hypercholesterolemia-associated mutations in a UK-based cohort: Development of an NGS-based method and comparison with multiplex PCR and oligonucleotide arrays.

Reiman A1, Pandey S2, Lloyd KL3, Dyer N3, Khan M4, Crockard M5, Latten MJ6, Watson T6, Cree I2, Grammatopoulos D7.

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Abstract

BACKGROUND:

Detection of disease-associated mutations in patients with familial hypercholesterolemia (FH) 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 LDLR have been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories.

METHODS:

Using primers targeting the LDLR, APOB and PCSK9, 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 PCR (Elucigene FH20), oligonucleotide arrays (Randox FH array) or the Illumina NGS platform.

RESULTS:

In this small cohort, the NGS 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 2 cases of mutation misclassification of the Elucigene FH20 multiplex PCR assay. A number of novel mutations not previously reported, were also identified by the NGS method.

CONCLUSIONS:

Ion Torrent-based NGS can deliver a suitable alternative for the molecular investigation of FH patients, especially when comprehensive mutation screening for rare or unknown mutations is required. KEYWORDSNGS, FH mutation, molecular screening, LDLR, apoB.