Impact of genetic variability on NADPH oxidase activity: an extensive genotype-phenotype assessment

<b>Introduction:</b> Oxidative stress is implicated in various diseases, and the NADPH oxidase enzyme complex (NOX) is a significant source of reactive oxygen species (ROS). Research linking genetic polymorphisms to enzyme activity has produced conflicting results. <b>Methods:</b> We aimed to establish a robust protocol to assess NOX activity <i>in vitro</i> under highly standardized conditions and correlate these measurements with genetic polymorphisms catalogued by the 1000 Human Genome Project and the HapMap Project. Lymphoblastoid cell lines (LCLs) served as a model system with samples from healthy participants from three Caucasian populations. NOX activity stimulated by phorbol 12-myristate 13-acetate was measured using chemiluminescence in 290 LCLs (198 in a training and 92 in a test set) through a series of multiply repeated measurements per LCL comprising in total over 1,500 NOX activity assessments. The association between NOX activity and single nucleotide polymorphisms (SNPs) in the NOX subunit genes <i>CYBA</i>, <i>CYBB</i>, <i>NCF1</i>, <i>NCF2</i>, and <i>NCF4</i> was subsequently examined. <b>Results:</b> Out of 651 valid polymorphic markers, 308 had a minor allele frequency of ≥ 5%, and 15 SNPs showed a statistically significant correlation with NOX activity in the training set. However, these 15 associations were not confirmed in the test set (all <i>p</i> ≥ 0.1). Additional analyses treating all 290 LCLs as a single cohort yielded three associations at <i>p</i> < 0.01, i.e. <i>CYBA</i> rs1017828, <i>NCF1</i> rs191081238, and <i>NCF4</i> rs4821544. However, statistical significance could not be called for any of these genetic markers upon adjustment for multiple testing, regardless whether a co-dominant, dominant or recessive allelic effect was assumed. <b>Conclusion:</b> Our results do not support a reproducible impact of common genetic diversity in NOX subunits on the enzyme activity in LCLs of subjects of Caucasian origin. This study represents the largest evaluation concerning relationships between NOX genetic variants and enzyme activity to date.