Genetic variants associated with the risk of NAFLD and NASH exhibit pleiotropic effects.^{1, 2} Specifically, the non-synonymous rs738409 C/G variant in PNPLA3 (patatin-like phospholipase domain-containing protein 3), which encodes I148 M protein isoforms with a significant effect on the severity of several liver-related outcomes,³ presents pleiotropic effects on cells that mediate the immune response.^{1, 2} Figure 1 discloses the laboratory measurements and/or haematological traits associated with variants in PNPLA3 extracted from different data sources.

The COVID-19 pandemic revealed that NAFLD and NASH, -either explained by the presence of overlapping co-morbidities such as obesity and type 2 diabetes or by the presence of the systemic inflammation associated with the chronic liver condition-, appear to be a risk factor for severe SARS-CoV-2 infection.⁴ Hence, it was postulated that variants that predispose to NAFLD may also indirectly condition the severity of the COVID-19 infection. This hypothesis motivated interesting candidate gene association studies. For example the UKBB (UK Biobank) dataset was initially used to develop a genetic risk score for NAFLD based on the weighted effect of variants involved in the accumulation of fat in the liver (PNPLA3-TM6SF2-MBOAT7-GCKR).⁵ Thus, leveraging this information, Valenti et al examined the impact of that NAFLD-genetic risk score on the risk of COVID-19,⁵ and detected a trend of protection against COVID-19 conferred by rs738409.⁵ In addition, the authors observed a trend towards differential gene expression levels of SARS-CoV-2 receptors (ACE2) in a cohort of 125 obese subjects.⁵

More recently, two studies from Europe highlighted the potential involvement of the rs738409 in modifying the severity of COVID-19, including hospitalization and death rate.^{6, 7} Grimaudo et al found a direct association between the rs738409 G-allele and severe COVID-19 outcomes among patients aged 65 years or less.⁶

On the other side, Innes et al reported a striking inverse association analysis between the rs738409 and COVID-19 severity outcomes in 1585 UKBB participants.⁷ The authors found that the rs738409-G allele was independently associated with a reduced risk of COVID-19 hospitalization and mortality; the protective effect remained significant after adjusting for major demographic and disease risk factors, including underlying metabolic and liver co-morbidities.⁷ In addition, Innes et al⁷ performed a meta-analysis of three independent data sources that explored the potential association between rs738409 and COVID-19. This study included data from the FinnGen study (a population-based biobank comprising 83 patients with COVID-19 hospital admission and 274 SARS-CoV-2–positive patients without hospital admission), the Geisinger Health System (n = 854 subjects of European Ancestry, comprising 165 individuals COVID-19 hospitalization vs 689 SARS-CoV-2–positive patients without hospital admission), and the study of Grimaudo et al (a total of 383 COVID-19 patients).⁶ The pooled analysis of the above-mentioned data sources showed that the presumed protective effect of the G- ‘NASH-risk allele' on the morbidity and mortality of COVID-19 could not be further validated.⁷ However, it appears that there was a trend for association with a reduced risk of COVID-19 hospitalization/severe disease that did not reach statistical significance.⁷

Likewise, Bianco et al found that the rs738409 G allele has a tendency not only to be associated with protection against COVID-19 but also it was associated with lower C-reactive protein levels despite higher ALT and lower albumin in severe COVID-19 patients of European ancestry.⁸ Table 1 shows a summary of current evidence of the putative association between rs738409 and COVID-19 outcomes.

These remarkable findings deserve several reflections before raising unequivocal conclusions of the putative role of rs738409 on the course of COVID-19, one concerning the sample size of the studies and another concerning the biological plausibility of the reported association.

Specifically, a note of caution must be added to the initial enthusiasm about the role of a PNPLA3-variant in protecting against severe COVID-19 because it might trigger unexpected clinical consequences, including changes in the course of clinical trials and disease management. The analysis of the presumed protective effect of the variant on COVID-19 mortality and morbidity was done on a sample without enough power to detect such association/s.⁷ To detect reproducible causal variants associated with complex diseases, a sample size with statistical power is critical to the robustness of the conclusion/s of the study. For instance the study of Innes et al involved a sample of 267 patients who died due to COVID-19 and 1318 who survived.⁷ Unfortunately, the statistical power for the sample size used in the additive model to infer the presumed protective effect is ~0.52 (considering the informed frequency of the rs738409 G-allele (20%), the COVID-19 death prevalence of ~3%, and the explained effect of ~25% lower odds of COVID-19 death).⁷ Thereby, the results of this meta-analysis need to be interpreted with caution until the presumed association be consistently reproduced in larger cohorts.

Yet, the reported association is not only clinically relevant, but it seems to be also plausible.

For that reason, we would like to highlight putative explanations based on the presence of broad and systemic biological effect/s of variants located in PNPLA3.

The analysis of genotype-phenotype associations shows that PNPLA3 presents several variants that modulate epigenetic modifications, including changes in DNA methylation and histones in human immune cell types. Specifically, the intron rs2294915 C/G/T (chr22:44340904) variant that is not only in high linkage disequilibrium with rs738409 (r² 0.85)⁹ but also modifies the expression of PNPLA3¹⁰ and SAMM50, is associated with the histone modification H3K4me1 in CD14⁺ monocytes (beta: −0.8994, P = 3.00e-14) from blood donors of the United Kingdom population.¹¹ In fact, Donati et al showed that the PNPLA3-rs2294915 variant influences the phenotypic expression of the rs7383409 in a bimodal fashion by acting on the PNPLA3 mRNA stability probably through a cis-expression quantitative trait locus.¹⁰

Finally, Phenome-wide association (PheWAS) analysis on data of the UK Biobank denotes not only beta positive ('risk') effects of rs738409 on liver-related traits but also beta negative ('protective') effects on many non-liver traits, including respiratory diseases and haematological-related traits, such as neutrophil and platelet count.² Of note, platelet count was associated with over fivefold enhanced risk of severe COVID-19 (OR, 5.1).¹² In addition, a higher neutrophil/monocytes ratio is another predictor of COVID19 worse outcome.

However, it is difficult to interpret how exactly the PNPLA3-rs738409 variant may differentially affect these traits in healthy individuals with and without COVID-19 infection. It is thus clear from the above discussion that there is a great opportunity for further genetic association studies. Epigenetic investigations based on the rs738409 can also be of help at identification of factors that may affect COVID-19 outcomes, including not only changes in cells of the immune system and platelets but also interactions with host lipid metabolism and SARS-CoV-2 life cycle.

Collectively, these observations might have several implications beyond the potential presumed protective effect of rs738409 on the course of COVID-19. For example the large and widely reproducible effect of rs738409 on the biology of NASH and fibrosis¹³ has positioned PNPLA3 as an attractive target for pharmacological intervention. The first in vivo experimental study examining the effect of antisense oligonucleotide (ASO)-mediated silencing of Pnpla3 in a knock-in mouse model in which the human PNPLA3-I148M mutation was introduced showed that Pnpla3 ASO therapy can improve all features of NAFLD, including liver fibrosis.¹⁴ Following advancements in humans were done as well. For example AZD2693 is a ligand-conjugated antisense therapy designed to inhibit the production of PNPLA3 protein. A clinical trial (ClinicalTrials.gov Identifier: NCT04483947) is recruiting volunteers to assess the safety and tolerability, pharmacokinetics and pharmacodynamics of AZD2693 in patients with NASH fibrosis and homozygous for the rs738409-G risk allele, in whom investigators should closely monitor COVID19 susceptibility.

Hence, the COVID-19 pandemic has brought attention to the importance of having a complete understanding not only of the PNPLA3 gene-attributed associations with liver-related traits but also a thorough understanding of the protein interactions, the active protein ligands, and most importantly, an accurate and comprehensive assessment of the impact of the variant pleiotropic effects.

与 NAFLD 和 NASH 风险相关的遗传变异表现出多效性。^1，2具体而言，非同义rs738409 C / G中的变体PNPLA3（patatin启动样磷脂酶结构域的蛋白3），其编码I148 M蛋白同种型与对几种肝相关的结果，其严重程度的显著效果^3所呈现对介导免疫反应的细胞的多效作用。^{1, 2}图 1 公开了与从不同数据源中提取的PNPLA3变异相关的实验室测量值和/或血液学特征。

COVID-19 大流行表明，NAFLD 和 NASH——无论是由于重叠的合并症如肥胖和 2 型糖尿病的存在，还是与慢性肝病相关的全身炎症的存在——似乎是一种风险严重 SARS-CoV-2 感染的因素。⁴因此，据推测，易患 NAFLD 的变异也可能间接影响 COVID-19 感染的严重程度。这一假设激发了有趣的候选基因关联研究。例如，UKBB（英国生物银行）数据集最初用于根据参与肝脏脂肪积累的变异的加权效应（PNPLA3 - TM6SF2 - MBOAT7 -GCKR )。⁵因此，利用这些信息，Valenti 等人检查了 NAFLD 遗传风险评分对 COVID-19 风险的影响，⁵并检测到 rs738409 赋予的针对 COVID-19 的保护趋势。⁵此外，作者在 125 名肥胖受试者的队列中观察到 SARS-CoV-2 受体 ( ACE2 )基因表达水平的差异趋势。⁵

最近，来自欧洲的两项研究强调了 rs738409 可能参与改变 COVID-19 的严重程度，包括住院率和死亡率。^{6, 7} Grimaudo 等人发现 rs738409 G 等位基因与 65 岁或以下患者的严重 COVID-19 结果之间存在直接关联。⁶

另一方面，Innes 等人报告了 1585 名 UKBB 参与者的 rs738409 和 COVID-19 严重程度结果之间惊人的反向关联分析。⁷作者发现 rs738409-G 等位基因与降低 COVID-19 住院和死亡风险独立相关；在调整主要人口统计学和疾病风险因素（包括潜在的代谢和肝脏合并症）后，保护作用仍然显着。⁷此外，Innes 等人⁷对三个独立数据源进行了荟萃分析，探讨了 rs738409 和 COVID-19 之间的潜在关联。本研究包括来自 FinnGen 研究（一个基于人群的生物库，包括 83 名 COVID-19 住院患者和 274 名未住院的 SARS-CoV-2 阳性患者）、Geisinger 卫生系统（n = 854 名欧洲血统受试者）的数据，包括 165 名 COVID-19 住院患者与 689 名未住院的 SARS-CoV-2 阳性患者），以及 Grimaudo 等人的研究（共 383 名 COVID-19 患者）。⁶上述数据来源的汇总分析表明，无法进一步验证 G-“NASH 风险等位基因”对 COVID-19 发病率和死亡率的假定保护作用。⁷然而，似乎存在与 COVID-19 住院/重症风险降低相关的趋势，但并未达到统计学显着性。⁷

同样，Bianco 等人发现 rs738409 G 等位基因不仅与针对 COVID-19 的保护有关，而且与较低的 C 反应蛋白水平有关，尽管重症 COVID-19 患者的 ALT 和白蛋白水平较低。欧洲血统。⁸表 1 总结了 rs738409 和 COVID-19 结果之间假定关联的当前证据。

在对 rs738409 在 COVID-19 过程中的假定作用提出明确结论之前，这些非凡的发现值得进行多次反思，一个是关于研究的样本量，另一个是关于所报告关联的生物学合理性。

具体来说，必须在最初对PNPLA3变体在预防严重 COVID-19 中的作用的热情中添加一个警告，因为它可能引发意外的临床后果，包括临床试验和疾病管理过程中的变化。该变异对 COVID-19 死亡率和发病率的假定保护作用的分析是对样本进行的，但没有足够的功效来检测这种关联。⁷为了检测与复杂疾病相关的可重复的因果变异，具有统计功效的样本量对于研究结论的稳健性至关重要。例如，Innes 等人的研究涉及 267 名因 COVID-19 死亡的患者和 1318 名幸存的患者样本。⁷不幸的是，加法模型中用于推断假定保护作用的样本量的统计功效为~0.52（考虑到 rs738409 G 等位基因的通知频率（20%），COVID-19 死亡率约为 3%，以及 COVID-19 死亡几率降低约 25% 的解释效应）。⁷因此，需要谨慎解释这项荟萃分析的结果，直到假定的关联在更大的队列中得到一致重现。

然而，所报告的关联不仅具有临床相关性，而且似乎也是合理的。

出于这个原因，我们想强调基于PNPLA3中变体的广泛和全身生物学效应的存在的推定解释。

基因型-表型关联的分析表明，PNPLA3呈现出几种调节表观遗传修饰的变体，包括人类免疫细胞类型中 DNA 甲基化和组蛋白的变化。具体来说，内含子 rs2294915 C/G/T (chr22:44340904) 变体不仅与 rs738409 (r ² 0.85) ⁹处于高度连锁不平衡，而且还修饰了 PNPLA3 ¹⁰和 SAMM50的表达，与组蛋白修饰 H3 相关在 来自英国人群的献血者的CD14 ⁺单核细胞（β：-0.8994，P = 3.00e-14）中。¹¹事实上，Donati 等人表明PNPLA3-rs2294915 变体可能通过顺式表达数量性状基因座影响PNPLA3 mRNA 稳定性，从而以双峰方式影响 rs7383409 的表型表达。¹⁰

最后，对英国生物银行数据的全表型关联 ( PheWAS ) 分析表明 rs738409 不仅对肝脏相关性状有 β 阳性（“风险”）影响，而且对许多非肝脏性状也有 β 阴性（“保护性”）影响，包括呼吸系统疾病和血液学相关特征，如中性粒细胞和血小板计数。²值得注意的是，血小板计数与严重 COVID-19 的风险增加超过五倍有关（OR，5.1）。¹²此外，较高的中性粒细胞/单核细胞比率是 COVID19 较差结果的另一个预测因素。

然而，很难解释PNPLA3 -rs738409 变体究竟如何对感染和未感染 COVID-19 的健康个体的这些特征产生不同的影响。因此，从上述讨论中可以清楚地看出，进一步的遗传关联研究有很大的机会。基于 rs738409 的表观遗传研究也有助于确定可能影响 COVID-19 结果的因素，不仅包括免疫系统和血小板的变化，还包括与宿主脂质代谢和 SARS-CoV-2 生命周期的相互作用.

总的来说，这些观察结果可能会产生一些影响，超出了 rs738409 对 COVID-19 病程的潜在保护作用。例如，rs738409 对 NASH 和纤维化生物学的巨大且可广泛重复的影响¹³已将 PNPLA3 定位为药物干预的有吸引力的目标。第一项体内实验研究检查了反义寡核苷酸 (ASO) 介导的Pnpla3沉默在引入人PNPLA3 -I148M 突变的敲入小鼠模型中的作用，结果表明 Pnpla3 ASO 疗法可以改善 NAFLD 的所有特征，包括肝纤维化。¹⁴人类的进步也随之而来。例如，AZD2693 是一种配体偶联的反义疗法，旨在抑制 PNPLA3 蛋白的产生。一项临床试验（ClinicalTrials.gov 标识符：NCT04483947）正在招募志愿者，以评估 AZD2693 在 NASH 纤维化和纯合子 rs738409-G 风险等位基因患者中的安全性和耐受性、药代动力学和药效学，研究人员应密切监测 COVID19。

因此，COVID-19 大流行引起了人们的注意，不仅要全面了解PNPLA3基因与肝脏相关性状的关联，还要全面了解蛋白质相互作用、活性蛋白质配体，最重要的是，对变异多效性影响的准确而全面的评估。