Inactivating Mutations in NPC1L1 and Protection from Coronary Heart Disease

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.) Ezetimibe, a drug that is commonly prescribed to reduce plasma levels of low-density lipoprotein (LDL) cholesterol, inhibits the function of the protein encoded by the Niemann– Pick C1-like 1 gene (NPC1L1).1 NPC1L1 protein, which is expressed in the small intestine and liver, functions as a transporter of dietary cholesterol from the gut lumen into intestinal enterocytes.2,3 Because of its ability to block sterol absorption by about 50%,4 ezetimibe lowers plasma LDL cholesterol levels by 15 to 20%.5 However, it is uncertain whether Copyright © 2014 Massachusetts Medical Society Address reprint requests to Dr. Sekar Kathiresan at the Cardiovascular Research Center and Center for Human Genetic Research, Massachusetts General Hospital, 185 Cambridge St., CPZN 5.252, Boston, MA 02114, or at [email protected]. The authors are listed in the Appendix. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. NIH Public Access Author Manuscript N Engl J Med. Author manuscript; available in PMC 2015 May 27. Published in final edited form as: N Engl J Med. 2014 November 27; 371(22): 2072–2082. doi:10.1056/NEJMoa1405386. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript inhibiting NPC1L1 — either through ezetimibe treatment or by other means — reduces the risk of clinical coronary heart disease.6 Naturally occurring DNA sequence variants in humans that affect the activity of one or more protein targets can be used to estimate the potential efficacy and toxicity of a drug targeting such proteins.7,8 Genomewide association studies have identified common DNA sequence variants in NPC1L1 associated with modest alterations in plasma LDL cholesterol levels.9 However, it is difficult to discern precisely how variants that are discovered through genomewide association studies affect the activity of a gene. In contrast, some DNA mutations that arise in the protein-coding sequence can completely inactivate a gene. Inactivating mutations can be single-base changes that introduce a stop codon and that lead to premature truncation of a protein (nonsense mutations), insertions or deletions (indels) of DNA that scramble the protein translation beyond the variant site (frameshift mutations), or point mutations at modification sites of the nascent pre– messenger RNA transcript that alter the splicing process10 (splice-site mutations). Because such mutations — which are variously termed protein-disruptive, protein-inactivating, lossof-function, or null — profoundly affect protein function, they are typically very rare in the population as a consequence of natural selection. We tested the hypothesis that protein-inactivating mutations in NPC1L1 reduce both the LDL cholesterol level and the risk of coronary heart disease. We sequenced the coding regions of NPC1L1 in a large number of persons, identified carriers of mutations that inactivate this gene, and determined whether persons who carry a heterozygous inactivating mutation had a lower LDL cholesterol level and a lower risk of coronary heart disease than noncarriers of these mutations.