Supplementary Material for: Functional Polymorphisms Regulate FOXO1 Transcript Expression and Contribute to the Risk and Symptom Severity of HDM-induced Allergic Rhinitis

Introduction
FOXO1 plays an important role in regulating immune processes that contribute to allergic inflammation, however, genetic variants influencing FOXO1 expression in AR pathogenesis remain unclear. This study aimed to investigate the functional effect of FOXO1 single nucleotide polymorphisms (SNPs) on AR development by performing genetic association and functional analysis studies.
Methods
This study belongs to a part of an ongoing Singapore/Malaysia cross-sectional genetics and epidemiological study (SMCSGES). We assessed the associations of FOXO1 transcript expression levels in peripheral blood mononuclear cells (PBMC) with AR phenotype, total nasal symptom score (TNSS), and SNP genotype in a sub-cohort of n=658 individuals from the SMCSGES population. Associations of FOXO1 SNPs with AR were assessed in a cohort of n=5072 individuals from the SMCSGES population. In vitro promoter luciferase assay was used to evaluate the effect of AR-associated SNPs on FOXO1 promoter activity.
Results
FOXO1 transcript expression in PBMC was significantly associated with the risk of AR (p<0.05) and TNSS among AR patients (p<0.0001). We identified a significant association between tag-SNPs rs9549246 and FOXO1 transcript expression in PBMC from the SMCSGES sub-cohort and the multi-ethnic eQTLGen consortium (false discovery rate-adjusted p<0.05). The minor allele “A” of tag-SNP rs9549246 was significantly associated with a higher risk of AR (p=0.04422, odds ratio=1.21, 95% confidence interval=1.01-1.45) in the SMCSGES genotyping cohort (n=5072). In vitro luciferase assay showed the minor allele “A” of rs35594717 (tagged by rs9549246) was significantly associated with a higher FOXO1 promoter activity (p<0.05).
Conclusion
FOXO1 transcript expression in PBMC has a strong association with the risk and symptom severity of AR. Genetic variants tagged by rs9549246 were shown to affect the expression of FOXO1 and contribute to the development of AR in the SMCSGES population.

引言
叉头框蛋白O1（FOXO1）在调控介导变应性炎症的免疫过程中发挥重要作用，但在变应性鼻炎（allergic rhinitis, AR）发病机制中影响FOXO1表达的遗传变异仍不明确。本研究旨在通过开展遗传关联与功能分析研究，探究FOXO1单核苷酸多态性（single nucleotide polymorphisms, SNPs）对AR发生发展的功能效应。

方法
本研究为正在进行的新加坡/马来西亚横断面遗传与流行病学研究（Singapore/Malaysia cross-sectional genetics and epidemiological study, SMCSGES）的一部分。我们从SMCSGES人群中选取n=658名个体组成亚队列，分析外周血单个核细胞（peripheral blood mononuclear cells, PBMC）中FOXO1转录本表达水平与AR表型、总鼻部症状评分（total nasal symptom score, TNSS）及单核苷酸多态性基因型的关联。同时，在SMCSGES人群中n=5072名个体组成的队列中，评估FOXO1 SNPs与AR的关联。本研究采用体外启动子荧光素酶报告基因实验，探究与AR相关的SNPs对FOXO1启动子活性的影响。

结果
PBMC中FOXO1转录本表达水平与AR发病风险显著相关（P<0.05），且在AR患者中与TNSS呈显著关联（P<0.0001）。我们在SMCSGES亚队列及多民族eQTLGen联盟（multi-ethnic eQTLGen consortium）的样本中，发现标签SNPs rs9549246与PBMC中FOXO1转录本表达水平存在显著关联（错误发现率（false discovery rate）校正后P<0.05）。在SMCSGES基因分型队列（n=5072）中，标签SNP rs9549246的次要等位基因“A”与更高的AR发病风险显著相关（P=0.04422，比值比（odds ratio, OR）=1.21，95%置信区间（95% confidence interval, 95%CI）=1.01~1.45）。体外荧光素酶实验结果显示，rs9549246标记的rs35594717的次要等位基因“A”可显著提升FOXO1启动子活性（P<0.05）。

结论
PBMC中FOXO1转录本表达水平与AR的发病风险及症状严重程度密切相关。在SMCSGES人群中，rs9549246标记的遗传变异可调控FOXO1表达，并参与AR的发生发展。