Now a team from the Université de Montréal, the Azrieli CHU Sainte-Justine Research Centre, and the Montreal Heart Institute has put that genetic support to its most rigorous test to date. Writing in Frontiers in Genetics, Tristan St-Laurent, Julien St-Pierre, Julien Quang Le Van, and Marc-André Legault revisited the association between ALOX5 and ALOX5AP variation and atherosclerotic cardiovascular disease using data from 378,962 participants in the UK Biobank, organized into a trans-ancestral meta-analysis spanning four genetically predicted ancestry groups. Their conclusion is blunt: when ancestry is adequately accounted for, the previously reported genetic associations simply do not replicate, and neither hypothesis-driven replication analyses nor agnostic association scans support a role for genetic variation in these two genes in cardiovascular risk.

The technical heart of the study lies in how the researchers handled a polymorphism that has confounded the field for twenty years. In the promoter region of ALOX5, a tandem repeat of the sequence 5′-GCGGGG-3′, known as the Sp1 motif, occurs in variable copy numbers ranging from one to eleven. A landmark 2004 study in the New England Journal of Medicine reported that people carrying non-5-repeat alleles had significantly greater carotid intima-media thickness, a measure of subclinical atherosclerosis, in a cohort of 470 participants from the Los Angeles Atherosclerosis Study. That finding became a cornerstone of the 5-LO hypothesis. But the distribution of Sp1 repeat counts varies dramatically across populations. The UK Biobank analysis confirmed that the most common total repeat count is eight in individuals of African ancestry versus ten in other groups, with the 5-repeat allele predominating in Europeans and deletion alleles being far more frequent in Africans. Because cardiovascular disease prevalence and mean carotid thickness also differ across ancestries, the authors argue that residual population stratification, the confounding of genetic variants with ancestry-linked disease risk, could have generated false positives in earlier studies that used the 5-repeat count as a universal reference point.

To genotype the tandem repeat at scale, the team relied on whole-genome sequencing calls from the UK Biobank’s DRAGEN pipeline, which achieves SNV and INDEL precision exceeding 99.8 percent against Genome in a Bottle standards. They then applied two association models: a direct replication of the original homozygous non-5-repeats coding, and an ancestry-specific dosage model in which copy number is coded relative to the most common allele within each ancestry group. The power calculations are striking. In the European ancestry group alone, the study had 80 percent power to detect a carotid thickness effect of roughly eight micrometers, while the original study reported mean differences exceeding 62 micrometers, meaning the new analysis had approximately 99.9 percent power to detect an effect fourfold smaller. For myocardial infarction, the model could detect an odds ratio of 1.30. Despite this power, the Sp1 repeat variant showed no statistically significant association with C-reactive protein, carotid thickness, LDL cholesterol, myocardial infarction, ischemic stroke, or revascularization procedures. The one nominally significant finding, an association with C-reactive protein in the African ancestry subgroup, did not survive correction for multiple testing and was absent from the trans-ancestral analysis.

There was, however, one biologically coherent signal that survived. Using the ancestry-specific dosage model, the researchers found a significant negative association between the number of Sp1 repeats and plasma omega-6 fatty acid levels. Arachidonic acid, an omega-6 fatty acid, is the primary substrate of 5-LO, so a reduction in enzyme activity would be expected to cause substrate accumulation. This association was directionally concordant across ancestries and consistent with prior nutrigenetic studies linking ALOX5 variants to eicosanoid production. In other words, the promoter polymorphism does appear to influence 5-LO activity, yet that influence does not translate into measurable differences in atherosclerosis or clinical cardiovascular events. The drug target may be real, but the genetic case that its variation drives disease is not.

The ALOX5AP story followed a similar arc. In 2004, an Icelandic team reported that a four-SNP haplotype called HapA in the gene encoding 5-LO activating protein was associated with myocardial infarction and stroke, a finding that was subsequently replicated in Scottish cohorts but contradicted by later meta-analyses of thirty studies finding no association with ischemic stroke. The Montreal team characterized substantial ancestral variability in HapA frequency, noting that one of its constituent SNPs is monomorphic in East Asian populations. Carriers of HapA under a dominant model showed no significantly increased risk of any cardiovascular outcome. A recessive model restricted to homozygous carriers, leveraging the full cohort of 378,962 participants, revealed a modest association with myocardial infarction in the European ancestry subgroup only, with an odds ratio of 1.12, but this did not remain significant after Bonferroni correction across the fourteen statistical models and outcomes assessed. HapA showed no association with omega-6 levels, unlike the ALOX5 promoter variant, further weakening the biological plausibility of the marginal signal.

The researchers then went beyond replication. They tested whether the variants behave differently in individuals at high genetic risk of coronary artery disease, using interaction terms with a CAD polygenic risk score, and found no effect heterogeneity. They examined 24,188 individuals of European ancestry with established atherosclerotic disease at baseline, of whom 5,862 experienced an incident event during follow-up, and found no association between either gene and incident events in this high-risk population. Regional association scans across the ALOX5 and ALOX5AP gene regions, padded by 100 kilobases and analyzed with the REGENIE mixed-effects model, identified five ALOX5 variants reaching the significance threshold for myocardial infarction in the discovery analysis, but three failed to replicate in the independent CARDIoGRAMplusC4D cohort and the remaining two were not present in that dataset, making them likely false positives.

The team also deployed a transcriptome-wide association study, or TWAS, to test whether genetically predicted expression of eighteen leukotriene pathway genes is associated with coronary artery disease. Using S-PrediXcan with expression prediction models derived from GTEx in whole blood, aorta, and coronary artery, and summary statistics from 113,937 individuals with CAD, none of the genes showed a significant association. A gene set enrichment analysis of the leukotriene pathway ranked by TWAS z-scores revealed no significant enrichment either, with a normalized enrichment score of 0.76 and a p-value of 0.77. Finally, rare variant association testing using the Genebass resource from 394,841 UK Biobank exomes and the All by All platform from 392,030 All of Us whole genomes, employing burden tests and SKAT, found no significant associations with any ASCVD-related phenotype.

The implications for drug development are considerable. Genetic validation has become an increasingly important predictor of clinical success for drug targets, and this study systematically dismantles the genetic support for 5-LO and its activating protein as targets in atherosclerotic cardiovascular disease. The authors are careful to note what their analysis does not rule out. It cannot exclude gene-gene interactions within the leukotriene pathway, nutrient-gene interactions involving arachidonic acid availability, or the possibility that inhibiting 5-LO redirects arachidonic acid toward clinically relevant alternate fates. Indeed, a clinical study of asthma patients treated with zileuton, a 5-LO inhibitor, observed increased spontaneous platelet aggregation, consistent with the hypothesis that blocking the enzyme pushes substrate into the cyclooxygenase pathway, generating thromboxane and prostaglandins instead of leukotrienes. The authors suggest that cysteinyl leukotriene receptors or enzymes downstream of 5-LO may be more viable targets, partly because they would spare 5-LO-mediated lipoxin production, which appears beneficial in the plaque environment.

There are also genuine limitations to what genetics can resolve here. The UK Biobank lacks circulating leukotriene measurements, so the team could not use direct molecular markers of pathway activity, relying instead on omega-6 as a proxy. The analysis excluded some ancestry groups not available through self-reporting, such as Middle Eastern populations. And 5-LO activity may be highly localized within the plaque itself, a context that current TWAS approaches, which rely on bulk gene expression atlases from cohorts without necessarily having ASCVD, lack the resolution to detect. The authors propose that future studies incorporating precise molecular measurements of leukotriene pathway products may provide more direct evidence on the impact of genetic variation in these genes. For now, the message is clear: the inflammatory biology of atherosclerosis remains a promising therapeutic frontier, as demonstrated by the success of IL-1β blockers and colchicine in randomized trials, but the specific genetic case for targeting 5-LO and its activating protein in cardiovascular disease has collapsed under the weight of a properly powered, ancestry-aware analysis.

Subject of Research: Revisiting the genetic support of leukotriene pathway drug targets in atherosclerotic cardiovascular disease

Article Title: Revisiting the genetic support of leukotriene pathway drug targets in atherosclerotic cardiovascular disease

Article References: St-Laurent, T., St-Pierre, J., Le Van, J. Q., & Legault, M.-A. (2026). Revisiting the genetic support of leukotriene pathway drug targets in atherosclerotic cardiovascular disease. Frontiers in Genetics, 17, Article 1812905. https://doi.org/10.3389/fgene.2026.1812905

Image Credits: AI Generated

DOI: 10.3389/fgene.2026.1812905

Keywords: Revisiting, genetic, support, leukotriene, pathway, drug, targets, atherosclerotic, cardiovascular, disease, scientific research