Profiling Peripheral Blood with an Optimized, Multiplexed, Single-cell Multiome Approach Supports an Insulin-driven Asthma Subtype

Abstract

RNA sequencing (RNA-seq) and the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) have become standard techniques for studying gene regulation in human populations. Single-cell (sc) "multiomic" genomic methodologies now enable researchers to dissect cellular heterogeneity while simultaneously measuring gene expression and chromatin accessibility within individual cells. However, single-cell approaches remain experimentally complex and cost-prohibitive, limiting their application in population studies, and motivating the development of new strategies for population-scale single-cell investigations. To this end, we have adapted and optimized a previous multiomic protocol, "Transcriptome, Epitope, and ATAC sequencing" (TEA-seq) through experimentation and simulation to incorporate sample multiplexing, thus resulting in our "multiplexed TEA-seq" (mTEA-seq) protocol. Using mTEA-seq, we sought to determine whether asthma that develops in conjunction with early-life elevated insulin levels might have an identifiable molecular signature. We studied samples from adult individuals (54 subjects, 272,003 cells) from the Tucson Childrens Respiratory Study (TCRS), a birth cohort phenotypically characterized over four decades, to identify unique molecular characteristics of blood cells from asthmatics who had high serum insulin levels at age 6. Using a Bayesian approach, we found striking sex-specific effects. Male asthmatic subjects with high insulin at age 6 displayed widespread immune transcriptional and epigenetic alterations into adulthood compared to male non-asthmatic subjects without elevated insulin at age 6. We also found that male non-asthmatics with early-life high insulin showed epigenetic perturbations in adulthood, but not transcriptional changes. The consistency of epigenetic signals between these two groups that had high insulin at age 6 was highly cell-type-specific. For example, CD14+ monocytes displayed broadly common insulin-associated chromatin remodeling regardless of asthma status, while NK cells exhibited unique patterns of insulin-associated epigenetic reprogramming depending on asthma status. Finally, genotyping performed directly from our single-cell data enabled cell type-specific cis-QTL mapping that suggested HLA-DQB1 and AHI as genes for future study in insulin-associated asthma. Our investigation of childhood insulin-associated asthma demonstrates a metabolically-driven alterations on immune cells persisting into adulthood, thus providing a molecular signature of this asthma subtype, and offering novel insights for disease prevention and therapeutic intervention.