key: cord-0702614-paepl1vs
authors: Kerget, Ferhan; Kerget, Buğra; Kahraman, Çiğdem Yüce; Araz, Ömer; Akgün, Metin; Uçar, Elif Yılmazel; Sağlam, Leyla
title: Evaluation of the relationship between pentraxin 3 (PTX3) rs2305619 (281A/G) and rs1840680 (1449A/G) polymorphisms and the clinical course of COVID‐19
date: 2021-07-31
journal: J Med Virol
DOI: 10.1002/jmv.27238
sha: 16738ef74c3b024350fff6c0cd1c5226beac4a6f
doc_id: 702614
cord_uid: paepl1vs

Macrophage activation syndrome (MAS) is one of the main causes of morbidity and mortality in patients with coronavirus disease 2019 (COVID‐19). This study aimed to investigate the relationship between the pentraxin 3 (PTX3) gene polymorphisms rs2305619 (281A/G) and rs1840680 (1449A/G) and the development of MAS in patients with COVID‐19. The study included a total of 94 patients aged 18–45 who were diagnosed as having COVID‐19 between June and December 2020. PTX3 281A/G and 1449A/G polymorphism frequencies were evaluated. PTX3 281A/G allele and genotype frequencies did not deviate from Hardy–Weinberg (HW) equilibrium in the MAS or non‐MAS group (χ (2): 0.049, df: 2, p = 0.976, χ (2): 0.430, df: 2, p = 0.806). PTX3 1449A/G allele and genotype frequencies deviated significantly from HW equilibrium in the non‐MAS group (χ (2): 6.794, df: 2, p = 0.033) but not in the MAS group (χ (2): 2.256, df: 2, p = 0.324). The AG genotype was significantly more frequent in the non‐MAS group, while the AA genotype was significantly more frequent in the MAS group (χ (2): 11.099, df: 2, p= 0.004). Analysis of the PTX3 1449A/G polymorphism showed that individuals with the GG genotype had higher serum PTX3 levels than those with the AA and AG genotypes (p = 0.001 for both). Analysis of the PTX3 1449A/G polymorphism in patients with COVID‐19 showed that those with the AG genotype were relatively more protected from MAS compared with individuals with the AA genotype. In addition, lower serum PTX3 levels are observed in patients carrying the A allele.

and macrophage activation syndrome (MAS). Both have been attributed to an overabundance of pro-inflammatory cytokines that causes endothelial dysfunction and damage to various vital organs, primarily the lungs. 3 The prognostic parameters most commonly used in COVID-19 include C-reactive protein (CRP), D-dimer, ferritin, leukopenia, fibrinogen, prothrombin time, and interleukin-6 (IL- 6) levels. 4, 5 Of the cytokines produced by active macrophages, IL-6, IL-1β, and tumor necrosis factor-α (TNF-α) are the main contributors to the development of MAS in COVID-19. 6 IL-6 induces hepatic synthesis of CRP and serum amyloid P, which are known as short pentraxins, whereas IL-1β and TNF-α induce synthesis of pentraxin 3 (PTX3), a long pentraxin. PTX3 can be synthesized by macrophages, monocytes, leukocytes, dendritic cells, adipocytes, endothelial cells, and smooth muscle cells. 7 Elevated PTX3 levels have been observed in bacterial, viral, and fungal lung infections, and this elevation was strongly associated with mortality. 8 Studies of the PTX3 rs2305619 (281A/G) and rs1840680 (1449A/G) gene polymorphisms have shown that patients with the AA genotype are more susceptible to pulmonary tuberculosis and aspergillosis. 9, 10 In a study conducted on patients with coronary artery disease, in which macrophages play an important role in the development and progression of atheromatous plaques, no relationship between 281A/G and 1449A/G polymorphisms and coronary artery disease was detected, and plasma PTX3 levels did not differ significantly between genotypes. 11 PTX3 is a recently discovered molecule and its mechanism of action is not yet clear. Although the data on plasma PTX3 levels in inflammatory diseases are consistent, single-nucleotide polymorphism (SNP) studies are lacking. Therefore, this study investigated the association between the PTX3 281A/G and 1449A/G polymorphisms and the development of MAS, which is associated with elevated proinflammatory cytokine levels and has high mortality, in patients with COVID-19.

The study included 94 patients between the ages of 18 and 45 years with no known comorbidities who were diagnosed and treated for COVID-19 at the Erzurum Regional Training and Research Hospital between June and December 2020. Of these, 46 patients were admitted to the intensive care unit due to MAS and 48 patients were treated in the COVID-19 ward and did not develop MAS or ARDS.

Patients who presented with symptoms such as fever, cough, dyspnea, malaise, and sudden loss of taste/smell and had a history of contact with a confirmed or suspected COVID-19 patient or a history of international travel in the past 2 weeks were evaluated by posterioranterior chest X-ray. Those with suspicious lesions were further examined with high-resolution thoracic computed tomography (CT). COVID-19 diagnosis was confirmed by SARS-CoV-2 real-time polymerase chain reaction (PCR) testing of nasopharyngeal swab samples.

The patients' history and laboratory results were reviewed for assessment of eligibility according to the following exclusion criteria: any comorbidity such as chronic obstructive pulmonary disease (COPD), diabetes, uncontrolled hypertension, coronary artery disease, and malignancy; history of infectious or inflammatory disease or invasive surgical procedures within the last month; and high fasting blood glucose. The presence of coronary artery disease, asthma, COPD, and diabetes was assessed through consultations with the cardiology, chest diseases, and internal medicine departments. Three patients with MAS were excluded due to abnormal fasting blood glucose levels.

Biochemical parameters including CRP, D-dimer, troponin-I, ferritin, liver, and kidney function tests, and hematological, coagulation, and arterial blood gas parameters were evaluated at admission and updated daily.

Fever was defined as an axillary temperature of 37.3°C or higher. Clinical and laboratory response was evaluated after 24 h. If an adequate response was not observed, a second 400 mg dose of tocilizumab was given.

After 15 min of semi-supine rest, blood samples were collected from an antecubital vein into ethylenediaminetetraacetic acid (EDTA) anticoagulant blood collection tubes. Troponin-I concentrations were measured by chemiluminescent immunoassay using an Immulite 2500 (Siemens Medical Solutions). IL-6 and PTX3 were measured by enzyme-linked immunosorbent assay (Elabscience human ELISA Kit).

2.4.1 | DNA isolation protocol DNA was isolated from blood collected in EDTA tubes using a QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol. DNA quality was measured using the NanoDrop (ND-1000, Thermo Fischer Scientific).

Allele-specific SNP Type™ assays were performed using a Fluidigm Flex Six™ Genotyping IFC (Fluidigm Corp.). Specific target amplification (STA) was performed to increase the initial number of molecular targets. Thermal cycling was run on a Bioer Gene Pro thermal cycler at 95°C for 15 min followed by 14 cycles of 95°C for 15 s and 60°C for 4 min. SNP Type Assay mixes and sample mixes were prepared according to the manufacturer's protocol. A dynamic array was loaded with 4 μl of each 10× assay mix and 5 μl of each sample mix, then placed in the IFC Controller HX (Fluidigm) to complete the loading process. The dynamic array was then placed in the BioMark system (Fluidigm) for thermal cycling and fluorescent image acquisition using the SNP type E Flex Six v1 protocol. Data were collected using the BioMark system's built-in software (Figure 1 ). Genotyping application, ROX passive reference, and SNP type-FAM and SNP type-HEX probe types were selected. Comparison of PTX3 1449 A/G genotypes between the groups is shown in Table 3 . AG genotype frequency was significantly higher in the non-MAS group, whereas AA genotype frequency was significantly higher in the MAS group (χ 2 : 11.099, df: 2, p = 0.004). There was no statistically significant difference between the MAS and non-MAS groups (p = 0.689). Comparison of PTX3 1449 A/G allele frequencies also showed no significant differences between the groups (p = 0.84, 0.53).

When serum PTX3 levels were analyzed according to PTX3

1449A/G genotype, we found that levels of PTX3 were significantly lower in patients with the AA and AG genotypes compared to patient GG genotype (p = 0.001, 0.001). In the statistical comparison between patients with AA and AG genotypes, there was no significant difference in PTX3 levels (p = 0.53) (Figure 2 ). When PTX3 levels were compared between COVID-19 patients with and without the A allele, it was observed that patients without the A allele had significantly higher PTX3 levels (p = 0.001).

In this study evaluating PTX3 281A/G and 1449A/G polymorphisms in COVID-19 patients with and without MAS, we observed no significant relationship between MAS and the 281A/G polymorphism.

However, for the 1449A/G polymorphism, the AG genotype was found to protect against progression to MAS while the AA genotype 19 In studies investigating PTX3 polymorphisms in pulmonary tuberculosis and aspergillosis patients, the 1449A/G polymorphism showed significant differences in both patient groups.

In addition, the AA genotype was found to be a risk factor for disease

The relationship between pentraxin 3 (PTX3) 1449A/G genotype frequency and plasma PTX3 levels This can be mainly attributed to the lack of a guiding analysis due to the low number of individuals with the AA genotype. The most important limitation of our study was that PTX3 1449A/G and 281A/G polymorphism levels were analyzed in a single race and in a limited population. However, the age range of the patients included in the study and ensuring they did not have any comorbidities were the main factors that limited our sample size. Larger multicenter studies are needed for our current findings to be generalized and utilized in the early diagnosis of MAS in clinical practice. As none of the patients in our study died during follow-up, it was not possible to evaluate serum PTX3 level or PTX3 1449A/G and 281A/G polymorphisms in terms of predicting mortality.

In conclusion, PTX3 is a recently characterized molecule in the long pentraxin family of acute-phase reactants that may guide treatment in the future. The clinical presentation of MAS that develops in COVID-19 can be diagnosed with follow-up, and these patients may not respond adequately to medical treatment. The results of our study on the PTX3 1449A/G polymorphism indicate that the AA genotype is more frequent among patients who develop MAS, while the AG genotype may be protective. With acquired immunity research and applications continuing at a brisk pace, early detection of polymorphisms may be life-saving for at-risk patients. Therefore, the results of our study may provide insight into the role of the newly discovered PTX3 in the future.

Pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (COVID-19): a review

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Interleukin-6 use in COVID-19 pneumonia related macrophage activation syndrome

The clinical implication of dynamic neutrophil to lymphocyte ratio and D-dimer in COVID-19: a retrospective study in Suzhou China

Hyperferritinemia in critically ill COVID-19 patients-Is ferritin the product of inflammation or a pathogenic mediator?

Are serum interleukin 6 and surfactant protein D levels associated with the clinical course of COVID-19?

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Antiviral activity of the long chain pentraxin PTX3 against influenza viruses

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Pentraxin 3 gene polymorphisms and pulmonary aspergillosis in chronic obstructive pulmonary disease patients

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Evaluation of the relationship between pentraxin 3 (PTX3) rs2305619 (281A/G) and rs1840680 (1449A/G) polymorphisms and the clinical course of COVID-19

The authors received no financial support for the research and/or authorship of this article.

The authors declare that there are no conflicts of interest. 

The data that support the findings of this study are available from the corresponding author upon reasonable request.

http://orcid.org/0000-0002-5160-4854