Table1_GWAS-significant loci and severe COVID-19: analysis of associations, link with thromboinflammation syndrome, gene-gene, and gene-environmental interactions.DOCX

ObjectiveThe aim of this study was to replicate associations of GWAS-significant loci with severe COVID-19 in the population of Central Russia, to investigate associations of the SNPs with thromboinflammation parameters, to analyze gene-gene and gene-environmental interactions.
Materials and MethodsDNA samples from 798 unrelated Caucasian subjects from Central Russia (199 hospitalized COVID-19 patients and 599 controls with a mild or asymptomatic course of COVID-19) were genotyped using probe-based polymerase chain reaction for 10 GWAS-significant SNPs: rs143334143 CCHCR1, rs111837807 CCHCR1, rs17078346 SLC6A20-LLZTFL1, rs17713054 SLC6A20-LLZTFL1, rs7949972 ELF5, rs61882275 ELF5, rs12585036 ATP11A, rs67579710 THBS3, THBS3-AS1, rs12610495 DPP9, rs9636867 IFNAR2.
ResultsSNP rs17713054 SLC6A20-LZTFL1 was associated with increased risk of severe COVID-19 in the entire group (risk allele A, OR = 1.78, 95% CI = 1.22–2.6, p = 0.003), obese individuals (OR = 2.31, 95% CI = 1.52–3.5, p = 0.0002, (pbonf = 0.0004)), patients with low fruit and vegetable intake (OR = 1.72, 95% CI = 1.15–2.58, p = 0.01, (pbonf = 0.02)), low physical activity (OR = 1.93, 95% CI = 1.26–2.94, p = 0.0035, (pbonf = 0.007)), and nonsmokers (OR = 1.65, 95% CI = 1.11–2.46, p = 0.02). This SNP correlated with increased BMI (p = 0.006) and worsened thrombodynamic parameters (maximum optical density of the formed clot, D (p = 0.02), delayed appearance of spontaneous clots, Tsp (p = 0.02), clot size 30 min after coagulation activation, CS (p = 0.036)). SNP rs17078346 SLC6A20-LZTFL1 was linked with increased BMI (p = 0.01) and severe COVID-19 in obese individuals (risk allele C, OR = 1.72, 95% CI = 1.15–2.58, p = 0.01, (pbonf = 0.02)). SNP rs12610495 DPP9 was associated with increased BMI (p = 0.01), severe COVID-19 in obese patients (risk allele G, OR = 1.48, 95% CI = 1.09–2.01, p = 0.01, (pbonf = 0.02)), and worsened thrombodynamic parameters (time to the start of clot growth, Tlag (p = 0.01)). For rs7949972 ELF5, a protective effect against severe COVID-19 was observed in non-obese patients (effect allele T, OR = 0.67, 95% CI = 0.47–0.95, p = 0.02, (pbonf = 0.04)), improving thrombodynamic parameters (CS (p = 0.02), stationary spatial clot growth rates, Vst (p = 0.02)). Finally, rs12585036 ATP11A exhibited a protective effect against severe COVID-19 in males (protective allele A, OR = 0.51, 95% CI = 0.32–0.83, p = 0.004). SNPs rs67579710 THBS3, THBS3-AS1, rs17713054 SLC6A20-LZTFL1, rs7949972 ELF5, rs9636867 IFNAR2—were involved in two or more of the most significant G×G interactions (pperm ≤ 0.01). The pairwise combination rs67579710 THBS3, THBS3-AS1 × rs17713054 SLC6A20-LZTFL1 was a priority in determining susceptibility to severe COVID-19 (it was included in four of the top five most significant SNP-SNP interaction models).
ConclusionOverall, this study represents a comprehensive molecular-genetic and bioinformatics analysis of the involvement of GWAS-significant loci in the molecular mechanisms of severe COVID-19, gene-gene and gene-environmental interactions, and provides evidence of their relationship with thromboinflammation parameters in patients hospitalized in intensive care units.

研究目的
本研究旨在俄罗斯中部人群中重复验证全基因组关联研究（Genome-Wide Association Study, GWAS）显著基因座与重症新型冠状病毒肺炎（COVID-19）的关联，探究单核苷酸多态性（Single Nucleotide Polymorphism, SNP）与血栓炎症（thromboinflammation）参数的关联，并分析基因-基因及基因-环境交互作用。

材料与方法
本研究纳入来自俄罗斯中部的798名无亲缘关系的高加索裔受试者，其中包括199名住院重症COVID-19患者，以及599名感染后表现为轻症或无症状的对照个体。采用基于探针的聚合酶链式反应对10个GWAS显著SNPs进行基因分型，具体位点如下：rs143334143 CCHCR1、rs111837807 CCHCR1、rs17078346 SLC6A20-LLZTFL1、rs17713054 SLC6A20-LLZTFL1、rs7949972 ELF5、rs61882275 ELF5、rs12585036 ATP11A、rs67579710 THBS3、THBS3-AS1、rs12610495 DPP9、rs9636867 IFNAR2。

结果
SNP rs17713054 SLC6A20-LLZTFL1与全队列人群重症COVID-19的发病风险升高显著相关（风险等位基因为A，比值比（Odds Ratio, OR）=1.78，95%置信区间（95% Confidence Interval, 95%CI）=1.22~2.6，P=0.003），该关联在肥胖个体（OR=2.31，95%CI=1.52~3.5，P=0.0002，Bonferroni校正P值（pbonf）=0.0004）、果蔬摄入不足人群（OR=1.72，95%CI=1.15~2.58，P=0.01，pbonf=0.02）、体力活动缺乏人群（OR=1.93，95%CI=1.26~2.94，P=0.0035，pbonf=0.007）及非吸烟者（OR=1.65，95%CI=1.11~2.46，P=0.02）中均成立。该SNP与体质量指数（Body Mass Index, BMI）升高（P=0.006）及恶化的血栓动力学参数相关，具体包括血凝块形成的最大光密度（D，P=0.02）、自发性血凝块出现延迟时间（Tsp，P=0.02）、凝血激活后30分钟血凝块大小（CS，P=0.036）。
SNP rs17078346 SLC6A20-LLZTFL1与BMI升高（P=0.01）及肥胖个体的重症COVID-19相关（风险等位基因为C，OR=1.72，95%CI=1.15~2.58，P=0.01，pbonf=0.02）。
SNP rs12610495 DPP9与BMI升高（P=0.01）、肥胖患者的重症COVID-19（风险等位基因为G，OR=1.48，95%CI=1.09~2.01，P=0.01，pbonf=0.02）及恶化的血栓动力学参数（血凝块生长起始时间Tlag，P=0.01）相关。
针对rs7949972 ELF5，在非肥胖患者中观察到其对重症COVID-19具有保护作用（效应等位基因为T，OR=0.67，95%CI=0.47~0.95，P=0.02，pbonf=0.04），同时可改善血栓动力学参数（CS，P=0.02；稳态空间血凝块生长速率Vst，P=0.02）。
最后，rs12585036 ATP11A在男性群体中对重症COVID-19表现出保护作用（保护等位基因为A，OR=0.51，95%CI=0.32~0.83，P=0.004）。
SNPs rs67579710 THBS3、THBS3-AS1、rs17713054 SLC6A20-LLZTFL1、rs7949972 ELF5及rs9636867 IFNAR2参与了2个及以上的显著基因-基因交互作用（置换检验P值（pperm）≤0.01）。其中，位点组合rs67579710 THBS3、THBS3-AS1 × rs17713054 SLC6A20-LLZTFL1是重症COVID-19易感性分析中的优先级交互模型，其被纳入前5个最显著的SNP-SNP交互模型中的4个。

结论
本研究对GWAS显著基因座在重症COVID-19分子机制、基因-基因及基因-环境交互作用中的参与情况进行了全面的分子遗传学与生物信息学分析，并为其与重症监护病房住院患者的血栓炎症参数的相关性提供了证据支持。