Inactivating mutations in NPC1L1 and protection from coronary heart disease

Abstract

Background: Ezetimibe lowers plasma levels of low-density lipoprotein (LDL) cholesterol by inhibiting the activity of the Niemann-Pick C1-like 1 (NPC1L1) protein. However, whether such inhibition reduces the risk of coronary heart disease is not known. Human mutations that inactivate a gene encoding a drug target can mimic the action of an inhibitory drug and thus can be used to infer potential effects of that drug.

Methods: We sequenced the exons of NPC1L1 in 7364 patients with coronary heart disease and in 14,728 controls without such disease who were of European, African, or South Asian ancestry. We identified carriers of inactivating mutations (nonsense, splice-site, or frameshift mutations). In addition, we genotyped a specific inactivating mutation (p.Arg406X) in 22,590 patients with coronary heart disease and in 68,412 controls. We tested the association between the presence of an inactivating mutation and both plasma lipid levels and the risk of coronary heart disease.

Results: With sequencing, we identified 15 distinct NPC1L1 inactivating mutations; approximately 1 in every 650 persons was a heterozygous carrier for 1 of these mutations. Heterozygous carriers of NPC1L1 inactivating mutations had a mean LDL cholesterol level that was 12 mg per deciliter (0.31 mmol per liter) lower than that in noncarriers (P=0.04). Carrier status was associated with a relative reduction of 53% in the risk of coronary heart disease (odds ratio for carriers, 0.47; 95% confidence interval, 0.25 to 0.87; P=0.008). In total, only 11 of 29,954 patients with coronary heart disease had an inactivating mutation (carrier frequency, 0.04%) in contrast to 71 of 83,140 controls (carrier frequency, 0.09%).

Conclusions: Naturally occurring mutations that disrupt NPC1L1 function were found to be associated with reduced plasma LDL cholesterol levels and a reduced risk of coronary heart disease. (Funded by the National Institutes of Health and others.).

Figure 1. Inactivating Mutations in NPC1L1 Identified in the Study

Black circles indicate individual mutations along with the effect expected to lead to NPC1L1 inactivation. Mutations p.L71RfsX50 and p.A296VfsX57 (red shading) are indels that shift the open reading frame and induce a premature termination codon after an additional 50 and 57 amino acids, respectively. Mutations c.1681+1G→A, c.2637+2T→G, and p.A1201V (c.3602C→T) (blue shading) alter the splicing process at sites of modification of the nascent pre-messenger RNA transcript (splice-site mutations). All other mutations (yellow shading) are single-nucleotide variants that introduce a termination codon. The locations of the three main extracellular domains, 13 transmembrane domains, and intracellular domains are based on data from Betters and Yu. NH₂ denotes the N-terminal at which protein translation is initiated, and COOH the C-terminal at which translation terminates.

Figure 2. Association between the Presence of Inactivating Mutations in NPC1L1 and LDL Cholesterol Levels, According to Genetic Ancestry

In each group of participants, we tested the association between the presence of inactivating mutations in NPC1L1 and plasma levels of low-density lipoprotein (LDL) cholesterol, after adjustment for age, sex, and study. The squares indicate the estimated adjusted difference in the LDL cholesterol level for carriers, as compared with noncarriers, in each ancestry group. The sizes of the squares are inversely proportional to the variance of the estimates. The diamonds indicate the combined results, based on a fixed-effects meta-analysis performed first within and then across ancestry groups. Participants from population-based studies — the Atherosclerosis Risk in Communities (ARIC) study, the Jackson Heart Study (JHS), and the Women’s Genome Health Study (WGHS) — and controls without coronary heart disease (CHD) from case–control studies were included in this analysis. To convert the values for cholesterol to millimoles per liter, multiply by 0.02586.