Nathan O. Stitziel

A general approach to single-nucleotide polymorphism discovery

Single-nucleotide polymorphisms (SNPs) are the most abundant form of human genetic variation and a resource for mapping complex genetic traits. The large volume of data produced by high-throughput sequencing projects is a rich and largely untapped source of SNPs (refs 2, 3, 4, 5). We present here a unified approach to the discovery of variations in genetic sequence data of arbitrary DNA sources. We propose to use the rapidly emerging genomic sequence as a template on which to layer often unmapped, fragmentary sequence data and to use base quality values to discern true allelic variations from sequencing errors. By taking advantage of the genomic sequence we are able to use simpler yet more accurate methods for sequence organization: fragment clustering, paralogue identification and multiple alignment. We analyse these sequences with a novel, Bayesian inference engine, POLYBAYES, to calculate the probability that a given site is polymorphic. Rigorous treatment of base quality permits completely automated evaluation of the full length of all sequences, without limitations on alignment depth. We demonstrate this approach by accurate SNP predictions in human ESTs aligned to finished and working-draft quality genomic sequences, a data set representative of the typical challenges of sequence-based SNP discovery.

Loss-of-Function Mutations in APOC3, Triglycerides, and Coronary Disease

BACKGROUND Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. METHODS We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. RESULTS An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G→A and IVS3+1G→T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P<1×10(-20)), and circulating levels of APOC3 in carriers were 46% lower than levels in noncarriers (P=8×10(-10)). The risk of coronary heart disease among 498 carriers of any rare APOC3 mutation was 40% lower than the risk among 110,472 noncarriers (odds ratio, 0.60; 95% confidence interval, 0.47 to 0.75; P=4×10(-6)). CONCLUSIONS Rare mutations that disrupt APOC3 function were associated with lower levels of plasma triglycerides and APOC3. Carriers of these mutations were found to have a reduced risk of coronary heart disease. (Funded by the National Heart, Lung, and Blood Institute and others.).

Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction

Summary Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance1,2. When MI occurs early in life, the role of inheritance is substantially greater1. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families3–8 whereas common variants at more than 45 loci have been associated with MI risk in the population9–15. Here, we evaluate the contribution of rare mutations to MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes where rare coding-sequence mutations were more frequent in cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare, damaging mutations (3.1% of cases versus 1.3% of controls) were at 2.4-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). This sequence-based estimate of the proportion of early MI cases due to LDLR mutations is remarkably similar to an estimate made more than 40 years ago using total cholesterol16. At apolipoprotein A-V (APOA5), carriers of rare nonsynonymous mutations (1.4% of cases versus 0.6% of controls) were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase15,17 and apolipoprotein C318,19. When combined, these observations suggest that, beyond LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk.

Exome sequencing and the genetic basis of complex traits

Shamil Sunyaev and colleagues present exome sequencing methods and their applications in studies to identify the genetic basis of human complex traits. They include analyses of the whole-exome sequences of 438 individuals from across several studies.

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.).

Genetic risk, coronary heart disease events, and the clinical benefit of statin therapy: an analysis of primary and secondary prevention trials.

BACKGROUND Genetic variants have been associated with the risk of coronary heart disease. In this study, we tested whether or not a composite of these variants could ascertain the risk of both incident and recurrent coronary heart disease events and identify those individuals who derive greater clinical benefit from statin therapy. METHODS A community-based cohort study (the Malmo Diet and Cancer Study) and four randomised controlled trials of both primary prevention (JUPITER and ASCOT) and secondary prevention (CARE and PROVE IT-TIMI 22) with statin therapy, comprising a total of 48,421 individuals and 3477 events, were included in these analyses. We studied the association of a genetic risk score based on 27 genetic variants with incident or recurrent coronary heart disease, adjusting for traditional clinical risk factors. We then investigated the relative and absolute risk reductions in coronary heart disease events with statin therapy stratified by genetic risk. We combined data from the different studies using a meta-analysis. FINDINGS When individuals were divided into low (quintile 1), intermediate (quintiles 2-4), and high (quintile 5) genetic risk categories, a significant gradient in risk for incident or recurrent coronary heart disease was shown. Compared with the low genetic risk category, the multivariable-adjusted hazard ratio for coronary heart disease for the intermediate genetic risk category was 1·34 (95% CI 1·22-1·47, p<0·0001) and that for the high genetic risk category was 1·72 (1·55-1·92, p<0·0001). In terms of the benefit of statin therapy in the four randomised trials, we noted a significant gradient (p=0·0277) of increasing relative risk reductions across the low (13%), intermediate (29%), and high (48%) genetic risk categories. Similarly, we noted greater absolute risk reductions in those individuals in higher genetic risk categories (p=0·0101), resulting in a roughly threefold decrease in the number needed to treat to prevent one coronary heart disease event in the primary prevention trials. Specifically, in the primary prevention trials, the number needed to treat to prevent one such event in 10 years was 66 in people at low genetic risk, 42 in those at intermediate genetic risk, and 25 in those at high genetic risk in JUPITER, and 57, 47, and 20, respectively, in ASCOT. INTERPRETATION A genetic risk score identified individuals at increased risk for both incident and recurrent coronary heart disease events. People with the highest burden of genetic risk derived the largest relative and absolute clinical benefit from statin therapy. FUNDING National Institutes of Health.

Distribution and Medical Impact of Loss-of-Function Variants in the Finnish Founder Population.

Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5–5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p<5×10−8) including splice variants in LPA that lowered plasma lipoprotein(a) levels (P = 1.5×10−117). Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease (OR = 0.84, P = 3×10−4), demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers.

Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease

A scavenger that protects the heart Coronary heart disease is a tale of two forms of plasma cholesterol. In contrast to the well-established effects of “bad” cholesterol (LDL-C), the role of “good” cholesterol (HDL-C) is mysterious. Elevated HDL-C correlates with a lower risk of heart disease, yet drugs that raise HDL-C levels do not reduce risk. Zanoni et al. found that some people with exceptionally high levels of HDL-C carry a rare sequence variant in the gene encoding the major HDL-C receptor, scavenger receptor BI. This variant destroys the receptors ability to take up HDL-C. Interestingly, people with this variant have a higher risk of heart disease despite having high levels of HDL-C. Science, this issue p. 1166 A human genetics study sheds light on how HDL (good) cholesterol protects against cardiovascular disease. Scavenger receptor BI (SR-BI) is the major receptor for high-density lipoprotein (HDL) cholesterol (HDL-C). In humans, high amounts of HDL-C in plasma are associated with a lower risk of coronary heart disease (CHD). Mice that have depleted Scarb1 (SR-BI knockout mice) have markedly elevated HDL-C levels but, paradoxically, increased atherosclerosis. The impact of SR-BI on HDL metabolism and CHD risk in humans remains unclear. Through targeted sequencing of coding regions of lipid-modifying genes in 328 individuals with extremely high plasma HDL-C levels, we identified a homozygote for a loss-of-function variant, in which leucine replaces proline 376 (P376L), in SCARB1, the gene encoding SR-BI. The P376L variant impairs posttranslational processing of SR-BI and abrogates selective HDL cholesterol uptake in transfected cells, in hepatocyte-like cells derived from induced pluripotent stem cells from the homozygous subject, and in mice. Large population-based studies revealed that subjects who are heterozygous carriers of the P376L variant have significantly increased levels of plasma HDL-C. P376L carriers have a profound HDL-related phenotype and an increased risk of CHD (odds ratio = 1.79, which is statistically significant).

Computational and statistical approaches to analyzing variants identified by exome sequencing

New sequencing technology has enabled the identification of thousands of single nucleotide polymorphisms in the exome, and many computational and statistical approaches to identify disease-association signals have emerged.

Meta-analysis identifies common and rare variants influencing blood pressure and overlapping with metabolic trait loci

Meta-analyses of association results for blood pressure using exome-centric single-variant and gene-based tests identified 31 new loci in a discovery stage among 146,562 individuals, with follow-up and meta-analysis in 180,726 additional individuals (total n = 327,288). These blood pressure–associated loci are enriched for known variants for cardiometabolic traits. Associations were also observed for the aggregation of rare and low-frequency missense variants in three genes, NPR1, DBH, and PTPMT1. In addition, blood pressure associations at 39 previously reported loci were confirmed. The identified variants implicate biological pathways related to cardiometabolic traits, vascular function, and development. Several new variants are inferred to have roles in transcription or as hubs in protein–protein interaction networks. Genetic risk scores constructed from the identified variants were strongly associated with coronary disease and myocardial infarction. This large collection of blood pressure–associated loci suggests new therapeutic strategies for hypertension, emphasizing a link with cardiometabolic risk.