Study Design

We conducted this study using data and DNA samples from 16 case–control studies and cohort studies. All study participants provided written informed consent for genetic studies. The first and last authors designed the study. The institutional review boards at the Broad Institute and each participating site approved the study protocols. The first and last authors vouch for the accuracy and completeness of the data and all analyses.

Study Participants

During the first phase of the study, we sequenced the 20 protein-coding exons in NPC1L1 in samples obtained from 22,092 participants from seven case–control studies and two prospective cohort studies (see Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). The case–control studies included the Exome Sequencing Project Early-Onset Myocardial Infarction (ESP-EOMI) study conducted by the National Heart, Lung, and Blood Institute,11 the Italian Atherosclerosis Thrombosis and Vascular Biology (ATVB) study,12 the Ottawa Heart Study (OHS),13 the Precocious Coronary Artery Disease (PROCARDIS) study,14 the Pakistan Risk of Myocardial Infarction Study (PROMIS),15 the Registre Gironi del COR (Gerona Heart Registry or REGICOR) study,16 and the Munich Myocardial Infarction (Munich-MI) study.17 The prospective cohort studies included the Atherosclerosis Risk in Communities (ARIC) study18 and the Jackson Heart Study (JHS).19

During the second phase of the study, we genotyped the most common inactivating mutation in NPC1L1 on the basis of data obtained during the sequencing phase (p.Arg406X) in nine independent sample sets from a total of 91,002 participants (Table S2 in the Supplementary Appendix). These nine sample sets were from participants in the ARIC study (participants who did not undergo sequencing), the Vanderbilt University Medical Center Biorepository (BioVU),20 the Genetics of Diabetes Audit and Research Tayside (GoDARTS) study,21 the German North and German South Coronary Artery Disease studies,22 the Mayo Vascular Diseases Biorepository (Mayo),23 PROCARDIS (participants who did not undergo sequencing), the Women's Genome Health Study (WGHS),24 and the Women's Health Initiative (WHI).25

Clinical Data

Data obtained for all the participants from both the sequencing and genotyping phases of the study included a medical history and laboratory assessment for cardiovascular risk factors, as described previously for each study. The participants were of African ancestry (2836 participants from ARIC, 2251 from JHS, and 455 from ESP-EOMI), South Asian ancestry (1951 participants from PROMIS), or European ancestry (all the other participants).

For each study cohort, available clinical data were used to define coronary heart disease. The definitions, which therefore varied from cohort to cohort, are provided in Tables S1 and S2 in the Supplementary Appendix.

Sequencing and Genotyping

Sequence data for NPC1L1 were extracted from exome sequences generated at the Broad Institute, the Human Genome Sequencing Center at Baylor College of Medicine, or the University of Washington with the use of protocols that are described in the Supplementary Appendix. Briefly, sequence reads were aligned to the human reference genome (build HG19), and the basic alignment files for sequenced samples were combined for the purpose of identifying variant positions. Single-nucleotide variants (SNVs) and indels were identified, and quality control procedures were applied to remove outlier samples and outlier variants, as described in the Supplementary Appendix.

For the purposes of this study, we defined inactivating mutations as any one of the following: SNVs leading to a stop codon substitution (nonsense mutations), SNVs occurring within two base pairs of an exon–intron boundary (splice-site mutations), or DNA insertions or deletions leading to a change in the reading frame and the introduction of a premature stop codon (frameshift mutations). The positions of nonsense, splice-site, and frameshift mutations were based on the complementary DNA reference sequence for NPC1L1 (NM_013389.2) with the ATG initiation codon, encoding methionine, numbered as residue 1 or p.Met1.

To obtain additional data for a particular nonsense mutation (p.Arg406X) observed from sequencing NPC1L1, we genotyped the variant site in additional samples using the HumanExome BeadChip Kit (Illumina), according to the manufacturer's recommended protocol. (See the Methods section in the Supplementary Appendix for details.)

Technical Validation of Sequencing and Genotyping

To assess the accuracy of next-generation sequencing methods, we performed Sanger sequencing on samples obtained from all participants who carried inactivating mutations in the ATVB study. To assess the accuracy of the genotyping of NPC1L1 p.Arg406X with the HumanExome BeadChip kit, we compared these genotypes with those derived from next-generation sequencing for a subset of samples.

Statistical Analysis

We first tested the association between NPC1L1 protein-inactivating mutations and plasma lipid levels. For participants who were receiving lipid-lowering therapy, we accounted for an average reduction in total cholesterol and LDL cholesterol levels of 20% and 30%, respectively,26 by adjusting the measured values accordingly. We did not adjust levels of high-density lipoprotein (HDL) cholesterol or triglycerides in these participants. Status with respect to the use of lipid-lowering medication was available for participants in ARIC, JHS, Munich-MI, PROCARDIS, REGICOR, and WGHS. When possible, we combined primary data for studies that included only one participant with an inactivating mutation with data for other studies involving participants of the same ancestry in order to create a larger data set. We performed regression analysis with a linear model that was adjusted for age and sex, along with an indicator variable for the study if applicable, to test for an association between the presence of inactivating mutations in NPC1L1 and levels of total cholesterol, LDL cholesterol, HDL cholesterol, and log-transformed triglyceride levels in each sample set. We combined results first within ancestry groups and then across ancestry groups, using fixed-effects meta-analyses.

We next tested for an association between protein-inactivating mutations in NPC1L1 and the risk of coronary heart disease. In each study, we estimated the odds ratio for disease among carriers of any NPC1L1 inactivating mutation, as compared with noncarriers. We then calculated the summary odds ratios and 95% confidence intervals for coronary heart disease among carriers, using a Mantel–Haenszel fixed-effects meta-analysis without continuity correction, a method that is robust with low (and even zero) counts and resultant odds ratios. A P value of less than 0.05 was considered to indicate statistical significance. The R software program (R Project for Statistical Computing) was used for all analyses.