Array-based resequencing for mutations causing familial hypercholesterolemia

Background: Familial hypercholesterolemia (FH) is a heterogeneous autosomal dominant disease with a prevalence of 1 in 500. To date, over 1200 unique pathogenic mutations have been identified in at least 3 genes. The large allelic and genetic heterogeneity of FH requires high-throughput, rapid, and affordable mutation detection technology to efficiently integrate molecular screening into clinical practice. We developed an array-based resequencing assay to facilitate genetic testing in FH patients. Methods and results: We designed a custom DNA resequencing array to detect mutations on all 3 FH-causing genes - LDL receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 gene (PCSK9) - and 290 known insertion/deletion mutations on LDLR. We verified FH array performance by analyzing 35 previously sequenced subjects (21 with point mutations, 2 insertions, 7 deletions, and 5 healthy controls) and blindly screening 125 FH patients. The average microarray call rate was 98.45% and the agreement between microarray and capillary sequencing was 99.99%. The FH array detected mutations by using automated software analysis, followed by manual review in 28 of the 30 subjects (pickup rate, 93.3%). In the blinded study, the FH array detected at least 1 mutation in 77.5% of patients clinically diagnosed with definite FH according to Simon Broome FH criteria and in 52.9% with probable FH diagnosis. Conclusions: The high-throughput FH resequencing array detects LDLR, APOB, and PCSK9 with high efficiency and accuracy and identifies disease-causing mutations. Thus, it facilitates large-scale screening of the heterogeneous FH populations.