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Background Pathogenic variants in the genes encoding the low-density lipoprotein receptor (LDLR), apolipoprotein B100 (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9)) are known to cause familial hypercholesterolemia (FH), a disease characterized by high LDL-C levels. We set out to investigate which proportion of patients with extremely elevated LDL-C levels, who were referred for molecular analysis, carry a mutation in any of the three FH genes or other, putative FH genes. Methods Targeted next-generation sequencing of 28 genes involved in lipid metabolism was performed in 1475 clinical FH patients with LDL-C levels >5mmol/L (corresponding with possible, probable and definite FH score according to the Dutch Lipid Clinic Network criteria) and triglyceride levels Results 14.3% of the 1475 patients with clinical FH were heterozygous carriers of a mutation in LDLR, APOB, or PCSK9. This number ranged from 8% in the lowest LDL-C group (5–5.99 mmol/L) to 56% in patients with the highest LDL-C levels (>8 mmol/L). Patients with LDLR, APOB, or PCSK9 variants had median LDL-C levels of 6.8 [5.7–7.9], 6.2 [5.1–7.0], and 6.4 [5.7–6.8] mmol/L [interquartile range], respectively. Mutation-negative FH patients had median LDL-C levels of 5.8 [5.2–6.5] mmol/L. Of the FH mutation-negative patients 7.4% had a (likely) pathogenic variant in the gene encoding lipoprotein lipase (LPL) and 1.3% in the apolipoprotein E (APOE) gene. The proportion of FH mutation-negative patients carrying an APOE variant increased from 1.2% in the lowest LDL-C group to 7.5% in patients with LDL-C >8 mmol/L. Mutation-positive FH patients were significantly younger (41.61±17.53 years vs 52.62±13.28 years P Figure 1 Conclusions A genetic defect in LDLR, APOB or PCSK9 is identified in only 14.3% of the patients with a clinical FH phenotype (defined as LDL-C >5 mmol/L) referred for molecular testing in the Netherlands. Mutations in LPL and APOE were found in a minor proportion of the FH mutation-negative patients. Our finding that a mutation is only found 56% of patients who present with LDL-C levels above 8 mmol/L indicates that either misclassification of FH and/or other genetic defects may be present in this group. Acknowledgement/Funding Grant [016.156.445] from The Netherlands Organisation for Scientific Research (NWO)

BACKGROUND Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused by mutations in 1 of 3 genes. In the 60% of patients who are mutation negative, we have recently shown that the clinical phenotype can be associated with an accumulation of common small-effect LDL cholesterol (LDL-C)-raising alleles by use of a 12–single nucleotide polymorphism (12-SNP) score. The aims of the study were to improve the selection of SNPs and replicate the results in additional samples. METHODS We used ROC curves to determine the optimum number of LDL-C SNPs. For replication analysis, we genotyped patients with a clinical diagnosis of FH from 6 countries for 6 LDL-C-associated alleles. We compared the weighted SNP score among patients with no confirmed mutation (FH/M–), those with a mutation (FH/M+), and controls from a UK population sample (WHII). RESULTS Increasing the number of SNPs to 33 did not improve the ability of the score to discriminate between FH/M– and controls, whereas sequential removal of SNPs with smaller effects/lower frequency showed that a weighted score of 6 SNPs performed as well as the 12-SNP score. Metaanalysis of the weighted 6-SNP score, on the basis of polymorphisms in CELSR2 (cadherin, EGF LAG 7-pass G-type receptor 2), APOB (apolipoprotein B), ABCG5/8 [ATP-binding cassette, sub-family G (WHITE), member 5/8], LDLR (low density lipoprotein receptor), and APOE (apolipoprotein E) loci, in the independent FH/M– cohorts showed a consistently higher score in comparison to the WHII population (P < 2.2 × 10−16). Modeling in individuals with a 6-SNP score in the top three-fourths of the score distribution indicated a >95% likelihood of a polygenic explanation of their increased LDL-C. CONCLUSIONS A 6-SNP LDL-C score consistently distinguishes FH/M– patients from healthy individuals. The hypercholesterolemia in 88% of mutation-negative patients is likely to have a polygenic basis.