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ORIGINAL ARTICLE
Year : 2022  |  Volume : 27  |  Issue : 6  |  Page : 718-722
 

To determine the genotyping of Fc-gamma receptor FCGR2A polymorphism as genetic susceptibility to neonatal sepsis: A study from a tertiary center of North India


1 Departments of Paediatric Surgery, IMS BHU, Varanasi, Uttar Pradesh, India
2 Department of Anatomy, Division of Genetic, MRU Lab, IMS BHU, Varanasi, Uttar Pradesh, India
3 Department of Anatomy, IMS BHU, Varanasi, Uttar Pradesh, India

Date of Submission14-Apr-2022
Date of Decision20-Aug-2022
Date of Acceptance12-Sep-2022
Date of Web Publication11-Nov-2022

Correspondence Address:
Sarita Chowdhary
Department of Paediatric Surgery, IMS BHU, Varanasi, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaps.jiaps_52_22

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   Abstract 


Background: Neonatal sepsis term is an infection of newborns <28 days of age. It is a common cause of death in developing countries. The receptor-gamma receptor FCGR2A has been shown to be associated with neonatal sepsis. It is an activating receptor found in many cell types such as monocytes, neutrophils, macrophages, platelets, and others. The receptor has a polymorphism (single-nucleotide polymorphism rs1801274) in its gene (FCGR2A) that encodes either a histidine (H) or arginine (R) at amino acid position 131. There are many studies showing the impact of these FCGR2A polymorphisms on sepsis. Our study aims to determine the prevalence of Fc-gamma receptor FCGR2A (rs1801274) polymorphism in neonatal sepsis and control in Eastern UP populations.
Patients and Methods: We conducted a cross-sectional descriptive study of 590 patients (310 healthy individuals and 280 sepsis patients) to determine polymorphisms in the CD32A coding region in neonates. All individuals were genotyped for a variant at position 131 of the FcγRIIA gene.
Discussion: In our study, the prevalence of FcγRIIa polymorphism is more in neonates with sepsis than in noninfected neonates. It was observed that the heterozygous allele (AG) were significantly increased in septic neonates when compared to the normal.
Conclusion: Our data indicate that FcγRIIA genotyping can be used as a marker of genetic susceptibility to sepsis.


Keywords: Neonatal sepsis, polymorphism, genotyping, single-nucleotide polymorphism


How to cite this article:
Chowdhary S, Sharma K, Ashish A, Yadav AK, Panigrahi P, Mishra A, Kumar D, Singh R. To determine the genotyping of Fc-gamma receptor FCGR2A polymorphism as genetic susceptibility to neonatal sepsis: A study from a tertiary center of North India. J Indian Assoc Pediatr Surg 2022;27:718-22

How to cite this URL:
Chowdhary S, Sharma K, Ashish A, Yadav AK, Panigrahi P, Mishra A, Kumar D, Singh R. To determine the genotyping of Fc-gamma receptor FCGR2A polymorphism as genetic susceptibility to neonatal sepsis: A study from a tertiary center of North India. J Indian Assoc Pediatr Surg [serial online] 2022 [cited 2022 Dec 7];27:718-22. Available from: https://www.jiaps.com/text.asp?2022/27/6/718/360964





   Introduction Top


Sepsis development is initiated by the recognition of one or more components of invading organisms such as Gram-negative endotoxins or secreted exotoxins that stimulate the local and systemic release of endogenous inflammatory mediators.[1] The production and release of these pro-inflammatory mediators can induce a systemic inflammatory response characteristic of the initial phase of sepsis.[2] Commonly used parameters for screening sepsis are total leukocyte count (TLC), immature-to-total neutrophil ratio, micro-erythrocyte sedimentation rate, and C-reactive protein (CRP).[3] The genetic variation in septic patients is a factor of research, as this factor is heterogeneous and affects individuals differently.[4] Genetic association with different immune profiles or clinical outcomes might help clinicians to diagnose sepsis and contribute to a better understanding of its pathophysiology.[5] Cellular cytotoxicity (ADCC), are functions depended on host Fc-gamma receptors (FcgRs) that bind the Fc portion of antibodies. Therefore a single-nucleotide polymorphisms (SNPs) of these receptors can affect immunoglobulin (Ig) G binding affinity. Accordingly, these SNPs can alter the ability of effector cells bearing FcγRs to facilitate Fc-mediated antibody functions.[6] There are many polymorphisms found on cells such as neutrophils, macrophages, and platelets. FcγRIIIa is less widely expressed than FcγRIIa. The receptor has a polymorphism (SNP rs1801274) in its gene (FCGR2A) that encodes either a histidine (H) or arginine (R) at amino acid position 131.[7]

The H allele is associated with higher affinity for IgG binding. It is the major FcγR found on natural killer cells, a key mediator of ADCC.[8] However, the study based on polymorphism and sepsis is variable.[9] Our study aims to determine FcγRs FCGR2A (rs1801274) polymorphism in neonatal sepsis and control in Eastern UP populations.

Study design

This was a cross-sectional descriptive study. Blood samples were collected at the neonatal intensive care unit of the Department of Paediatric Surgery, Sir Sunderlal Hospital, Institute of Medical Sciences, Banaras Hindu University (2018–2020), who exhibited clinical signs and symptoms of sepsis (that is tachycardia or bradycardia, increased or decreased respiratory rate, and hypothermia or hyperthermia) at the time of admission or who developed sepsis during their hospital stay according to the inclusion criteria. All participants were from Eastern UP and Bihar and of similar genetic background. The study was approved by the Institutional Medical Ethical Committee (No. Dean/2019/EC/1023) on January 18, 2019, and all participants provided their consent. The control group consisted of 310 proven healthy controls.

DNA extraction and genotyping

Five hundred and ninety samples were collected which include 310 samples from healthy individuals and 280 samples from patients with sepsis. Genomic DNA was extracted from leukocytes using a standard PureLink Genomic DNA Mini Kit-Thermo Fisher Scientific Cat No. K1820-01, LOT No. 1947948, and manual salting-out manual method.[9] DNA was diluted in water to a final concentration of 10 ng/μL per reaction. Moreover, mutation tests were performed using the TaqMan® (Invitrogen incorporating ready-made labeled SNP probes utilizing fluorescein-amide Applied Biosystems) SNP Genotyping Assay C9077561-20 LOT No. 4009825 for the polymorphism rs1801274 [Table 1], which detects the FcγRIIAR/H131 allele. The reaction was optimized for a total of 3 μL genomic DNA mixed with 6.25 μL TaqMan Universal Master Mix and 0.312 μL TaqMan SNP Genotyping Assay Mix. After an initial step at 95°C for 10 min, amplification was performed using 40 cycles of denaturation (92°C, 15 s), annealing (60°C, 1 min), and extension (60°C, 1 min). The amplification of the DNA for Taqman Assay was done using Real time polymerase chain reaction ( Applied Biosystems, Step one plus, USA)as per the protocol given by Taqman SNP genotyping assay kit (Applied Biosystems, Thermo Fisher Scientific, USA).
Table 1: Main characteristics of product details

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Statistical analysis

Allele discrimination was done using a 7500 fast RT-PCR system (version 2.3). Endpoint reads on the RT-PCR instrument were subsequently plotted by the instrument analysis software that makes allowances for autocalling genotypes, thereby minimizing manual intervention. Plotting is done generally by allele 1 (VIC® dye) on the X-axis and allele 2 (FAM dye) on the Y-axis [[Figure 1]; https://tools.thermofisher.com/content/sfs/manuals/TaqMan_SNP_Genotyping_Assays_man.Pdf]. After each run, the genotyped data were reviewed by two independent examiners who were blind to the all-participant status of the samples. The genotyped data were then moved to Excel sheets to be computed and statistically analyzed using the SPSS software program (IBM Corp. Released 2020. IBM SPSS Statistics for Windows, version 27.0. Armonk, NY, USA: IBM Corp). Qualitative variables were compared using Chi-square and Fisher's exact tests. Each group was tested separately for Hardy–Weinberg equilibrium using the Chi-square test. Allele carriage corresponds to the number of people carrying at least a single copy of a particular allele, whereas allelic frequency equates the number of appearances of a specific test allele divided by the overall count of alleles in the group.
Figure 1: Diagram represents the output file of allele discrimination software plot and multicomponent plot of FCGR2A (rs1801274). (a) Allelic discrimination plot: blue color represents the sample with FAM homozygote allele 2/allele 2 genotyping, green represents the sample with VIC and FAM heterozygote allele 1/allele 2 genotyping, orange color represents the sample with VIC homozygote genotyping, and X represents the undetermined samples. (b) The CG genotyping participants (carrying the A and G alleles) (heterozygote) among the sepsis subpopulation. RFU: Relative fluorescence unit, VIC: Victoria green fluorescent Protein, FAM: Fluorescein Amide

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Biochemical profile

Analysis of the patients blood for heamalogical and biochemical profile was done immediately using a centrifuge and stored at 20°C until processing of biochemical parameters. Fasting plasma glucose (enzymatic colorimetric method), hemoglobin, TLC, platelet (hematology analyzer), sodium, potassium, chloride, urea, and creatinine (free and total) were measured by chemiluminescence immunoassay analyzer in groups. The mean hemoglobin, TLC, platelet count, glucose, potassium, and chloride were comparable in the sepsis group (P > 0.05).


   Results Top


A total of 590 neonates were included in our study. Two hundred and eighty were neonates with sepsis and 310 were healthy neonates. Genotyping of all individuals included in the study was done.

Results were validated by randomly choosing samples for DNA sequencing. Three samples were analyzed for good results. All genotyping results matched perfectly and were aligned with the Hardy–Weinberg equilibrium.

The frequencies of the nucleotide of FcGR2A gene is elicited in [Table 2]. The allele frequencies of FcGR2A gene is summarised [Table 3].
Table 2: Nucleotide frequencies of the Fc-gamma receptor 2A rs1801274 polymorphism

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Table 3: Genotypic Frequency distribution ( Allele:Homozygous and heterozygous)of Fc-gamma 2A receptor polymorphism in neonates with sepsis and normal individuals

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Genotypic and allelic CD32A frequencies have a significant difference among the sepsis group than the healthy group. As per the data observed since the allele A has a higher odd's ratio ; the neonates having the Allele A are at a higher risk of developing sepsis than those neonates who have the allele G (P =0.0092). Allele AA is significantly more in sepsis neonates than homozygous GG, which is significantly more in healthy neonates. Allele AG (heterozygous) is nearly the same prevalence in both the groups of neonates.


   Discussion Top


There are many genetic variations observed in sepsis patients. Most of them concentrated on the components of innate immunity and the coagulation system of an individual. Cytokines, chemokines, and intracellular receptors have been studied for sepsis.[10] Gene products get altered due to SNP (single nitrogenous base) exchanged.[11] SNPs are the most commonly encountered polymorphism. Its incidence is approximately 1:1000 base pairs and commonly found is substitution of cytosine for thymine (C > T). It is estimated that 10% of all SNPs in the human genome are functional.[12] Several studies have investigated multiple SNPs from multiple genes with the hope of identifying biomarkers in complex diseases. The understanding of genetic polymorphism in population would facilitate understating the pathway of inflammatory process and thus help in the diagnosis and prognosis of the neonates with sepsis. Indeed, there is a requirement of differentiating excessive inflammation from sepsis in critical care medicine.[13] Unnecessary antibiotics are given in suspicious risk and death in proper confirmation of sepsis.[14] This might lead to antibiotic-resistant bacteria. Previously, FcγRIIA (CD32A) polymorphisms have been described in cases of infections, inflammation, and autoimmune diseases.[7] Examples include systemic lupus erythematosus, sickle cell disease, meningococcemia in children, malaria, human immunodeficiency virus infection, and Epstein–Barr virus infection.[8],[10] Homozygosity for FcγRIIA-H131 was associated with a higher risk of pneumococcal community-acquired pneumonia,[15] and neutrophils from participants homozygous for FcγRIIA-R131 exhibited a significantly reduced uptake of opsonized pneumococci, group B streptococci, Neisseria, and staphylococci compared with FcγRIIA-H131 cells.[16] We aimed to identify a FcγRIIA polymorphism to be more prevalent in sepsis neonates as compared to healthy neonates. The presence of allele A in the FcγRIIA gene was associated with an increased risk of sepsis in the study. Because FcγRIIA is an immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor, and the R131 polymorphism has a lower affinity for IgG2, this polymorphism might produce a weaker inflammatory response than the H131 genotype. However, many previous studies indicated that ITAM-bearing receptors also trigger inhibitory signals under special conditions; thus, it is very difficult to predict the signaling mechanisms elicited by FcγRIIA polymorphisms during sepsis.[16] Moreover, FcγRIIA binds to CRP, an acute-phase protein that recognizes pathogenic microbes and damaged cells, activates complement, and promotes the clearance of apoptotic cells.[9],[17] However, its mechanisms of action, including Fc receptor biology, are largely unknown. CRP exists in conformationally distinct forms, which explain its various functions. CRP-FcγRIIA interactions mediate potent antineutrophil and antiplatelet adhesion functions that limit inflammation and thrombosis. In addition, some FcγRIIA-mediated responses triggered by CRP are allele specific.


   Conclusion Top


Genetic polymorphism in neonates is associated with altered susceptibility to neonatal sepsis and thus it will identify the neonates susceptible to sepsis. This will allow more focused treatment. It will avoid unnecessary or detrimental drug administration. Furthermore, studies are needed to clarify why the R131 polymorphism is more prevalent in septic neonates.

Limitation

This study is descriptive and thus needs more analytic study to prove genetic polymorphism as a good and early prognostic marker for neonatal sepsis.

Authors' contribution

KS helped in formulating the study design, PP has done statistics, AC and RS have done laboratory research works, and DK and AM have done a literature search.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Machado JR, Soave DF, da Silva MV, de Menezes LB, Etchebehere RM, Monteiro ML, et al. Neonatal sepsis and inflammatory mediators. Mediators Inflamm 2014;2014:269681.4.  Back to cited text no. 1
    
2.
Rowe MI, Rowe SA. The last fifty years of neonatal surgical management. Am J Surg 2000;180:345-52.  Back to cited text no. 2
    
3.
Lin GC, Küng E, Smajlhodzic M, Domazet S, Friedl HP, Angerer J, et al. Directed transport of CRP across in vitro models of the blood-saliva barrier strengthens the feasibility of salivary CRP as biomarker for neonatal sepsis. Pharmaceutics 2021;13:256.7.  Back to cited text no. 3
    
4.
Iskander KN, Osuchowski MF, Stearns-Kurosawa DJ, Kurosawa S, Stepien D, Valentine C, et al. Sepsis: Multiple abnormalities, heterogeneous responses, and evolving understanding. Physiol Rev 2013;93:1247-88.  Back to cited text no. 4
    
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Namath A, Patterson AJ. Genetic polymorphisms in sepsis. Crit Care Nurs Clin North Am 2011;23:181-202.  Back to cited text no. 5
    
6.
van Erp EA, Luytjes W, Ferwerda G, van Kasteren PB. Fc-Mediated antibody effector functions during respiratory syncytial virus infection and disease. Front Immunol 2019;10:548.  Back to cited text no. 6
    
7.
Amiah MA, Ouattara A, Okou DT, N'Guetta SA, Yavo W. Polymorphisms in Fc gamma receptors and susceptibility to malaria in an endemic population. Front Immunol 2020;11:561142.  Back to cited text no. 7
    
8.
Castro-Dopico T, Clatworthy MR. IgG and Fcγ receptors in intestinal immunity and inflammation. Front Immunol 2019;10:805.  Back to cited text no. 8
    
9.
Beppler J, Koehler-Santos P, Pasqualim G, Matte U, Alho CS, Dias FS, et al. Fc gamma receptor IIA (CD32A) R131 polymorphism as a marker of genetic susceptibility to sepsis. Inflammation 2016;39:518-25.  Back to cited text no. 9
    
10.
Chen J, Wei H. Immune intervention in sepsis. Front Pharmacol 2021;12:718089.  Back to cited text no. 10
    
11.
Wang X, Tomso DJ, Liu X, Bell DA. Single nucleotide polymorphism in transcriptional regulatory regions and expression of environmentally responsive genes. Toxicol Appl Pharmacol 2005;207:84-90.  Back to cited text no. 11
    
12.
Neininger K, Marschall T, Helms V. SNP and indel frequencies at transcription start sites and at canonical and alternative translation initiation sites in the human genome. PLoS One 2019;14:e0214816.  Back to cited text no. 12
    
13.
Vincent JL. The clinical challenge of sepsis identification and monitoring. PLoS Med 2016;13:e1002022.  Back to cited text no. 13
    
14.
Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA 2016;315:801-10.  Back to cited text no. 14
    
15.
Shankar-Hari M, Phillips GS, Levy ML, Seymour CW, Liu VX, Deutschman CS, et al. Developing a new definition and assessing new clinical criteria for septic shock: For the third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA 2016;315:775-87.  Back to cited text no. 15
    
16.
Pinheiro da Silva F, Aloulou M, Benhamou M, Monteiro RC. Inhibitory ITAMs: A matter of life and death. Trends Immunol 2008;29:366-73.  Back to cited text no. 16
    
17.
Wang Y, Jönsson F. Expression, role, and regulation of neutrophil Fcγ receptors. Front Immunol 2019;10:1958.  Back to cited text no. 17
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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