Whole exome sequencing of familial hypercholesterolaemia patients negative for LDLR/APOB/PCSK9 mutations

Abstract

Background: Familial hypercholesterolaemia (FH) is an autosomal dominant disease of lipid metabolism, which leads to early coronary heart disease. Mutations in LDLR, APOB and PCSK9 can be detected in 80% of definite FH (DFH) patients. This study aimed to identify novel FH-causing genetic variants in patients with no detectable mutation.

Methods and results: Exomes of 125 unrelated DFH patients were sequenced, as part of the UK10K project. First, analysis of known FH genes identified 23 LDLR and two APOB mutations, and patients with explained causes of FH were excluded from further analysis. Second, common and rare variants in genes associated with low-density lipoprotein cholesterol (LDL-C) levels in genome-wide association study (GWAS) meta-analysis were examined. There was no clear rare variant association in LDL-C GWAS hits; however, there were 29 patients with a high LDL-C SNP score suggestive of polygenic hypercholesterolaemia. Finally, a gene-based burden test for an excess of rare (frequency <0.005) or novel variants in cases versus 1926 controls was performed, with variants with an unlikely functional effect (intronic, synonymous) filtered out.

Conclusions: No major novel locus for FH was detected, with no gene having a functional variant in more than three patients; however, an excess of novel variants was found in 18 genes, of which the strongest candidates included CH25H and INSIG2 (p<4.3×10(-4) and p<3.7×10(-3), respectively). This suggests that the genetic cause of FH in these unexplained cases is likely to be very heterogeneous, which complicates the diagnostic and novel gene discovery process.

Keywords: Cardiovascular Medicine; Diagnosis; Genetics; Lipid Disorders.

Figure 1

Novel familial hypercholesterolaemia (FH) gene discovery pipeline. (A) To increase the chance of detecting true FH-causing variants with a strong effect and reduce the noise, samples with a mutation in LDLR or APOB (apart from novel APOB variants of unknown effect) or those with a high low-density lipoprotein cholesterol (LDL-C) gene score were removed from the analysis. The remaining variants were filtered by their frequency and functional effect and compared against controls. Genes with more than four novel functional variants in controls or genes located on the X chromosome were filtered out to enhance the power of the test. The remaining variants were manually assessed and false positive calls were removed. (B) Comparison of the LDL-C SNPs score among the WHII control population (n=3020), FH mutation positive individuals (n=21) and FH mutation negative individuals (n=83) in a standard boxplot (the minimum, lower quartile, median, upper quartile and maximum). The overall difference between the groups was highly significant (ANOVA, p<2.2×10⁻¹⁶). Dashed line indicates the top decile cut-off for the WHII cohort (=1.16). A gene score was not attainable for 16 samples due to a poor DNA quality and insufficient concentration, which resulted in incomplete genotype data. (C) Schematic representation of the intronless CH25H gene and the localisation of novel variants identified in the FH cohort (in boxes). CH25H encodes an enzyme, cholesterol 25-hydroxylase, known to be spanning the endoplasmic reticulum membrane, with two domains (including the N-terminal) located outside of the membrane (in light grey), three 20 amino acid long transmembrane regions and two domains positioned inside the membrane, which contain three His boxes, essential for the catalytic activity of the enzyme.