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Original Article

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Blood Res 2020; 55(4):

Published online December 31, 2020

https://doi.org/10.5045/br.2020.2020009

© The Korean Society of Hematology

Prognostic value of TNF-a-308 and IFN-g-874 single nucleotide polymorphisms and their plasma levels in patients with aplastic anemia

Saurabh Shukla, Anil Kumar Tripathi, Shailendra Prasad Verma, Nidhi Awasthi

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Department of Clinical Hematology, King George’s Medical University, Lucknow, India

Correspondence to : Anil Kumar Tripathi, M.D.
Department of Clinical Hematology, King George’s Medical University, Shamina Road, Lucknow 226003, India
E-mail: aktkgmu@gmail.com

Received: January 15, 2020; Revised: August 20, 2020; Accepted: September 25, 2020

Abstract

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Background
Aplastic anemia (AA), an unusual hematological disease, is characterized by hypoplasia of the bone marrow and failure to form blood cells of all three lineages resulting in pancytopenia. This study aimed to investigate TNF-a-308 and IFN-g-874 gene polymorphisms and their respective plasma protein levels in patients with AA and healthy controls.
Methods
Two hundred and forty individuals were included in this study; the case group comprised 120 AA patients, while 120 healthy individuals served as controls. Genotyping was performed using the PCR-restriction length fragment polymorphism method and TNF-a-308 and IFN-g-874 plasma levels were evaluated using an ELISA kit.
Results
There was a significantly higher prevalence of the IFN-g-874 genotype in patients with AA than in healthy controls, while the TNF-a-308 genotype was associated with lower risk of developing AA. Furthermore, the levels of both TNF-a-308 and IFN-g-874 were higher in the plasma of AA patients.
Conclusion
Our findings suggest that the IFN-g-874 genotype may be a greater risk factor in the causation of AA, whereas the TNF-a-308 genotype has a protective role in the North Indian population.

Keywords Aplastic anemia, TNF-a-308, IFN-g-874, Polymorphism, PCR-RFLP, ELISA

Article

Original Article

Blood Res 2020; 55(4): 193-199

Published online December 31, 2020 https://doi.org/10.5045/br.2020.2020009

Copyright © The Korean Society of Hematology.

Prognostic value of TNF-a-308 and IFN-g-874 single nucleotide polymorphisms and their plasma levels in patients with aplastic anemia

Saurabh Shukla, Anil Kumar Tripathi, Shailendra Prasad Verma, Nidhi Awasthi

Department of Clinical Hematology, King George’s Medical University, Lucknow, India

Correspondence to:Anil Kumar Tripathi, M.D.
Department of Clinical Hematology, King George’s Medical University, Shamina Road, Lucknow 226003, India
E-mail: aktkgmu@gmail.com

Received: January 15, 2020; Revised: August 20, 2020; Accepted: September 25, 2020

Abstract

Background
Aplastic anemia (AA), an unusual hematological disease, is characterized by hypoplasia of the bone marrow and failure to form blood cells of all three lineages resulting in pancytopenia. This study aimed to investigate TNF-a-308 and IFN-g-874 gene polymorphisms and their respective plasma protein levels in patients with AA and healthy controls.
Methods
Two hundred and forty individuals were included in this study; the case group comprised 120 AA patients, while 120 healthy individuals served as controls. Genotyping was performed using the PCR-restriction length fragment polymorphism method and TNF-a-308 and IFN-g-874 plasma levels were evaluated using an ELISA kit.
Results
There was a significantly higher prevalence of the IFN-g-874 genotype in patients with AA than in healthy controls, while the TNF-a-308 genotype was associated with lower risk of developing AA. Furthermore, the levels of both TNF-a-308 and IFN-g-874 were higher in the plasma of AA patients.
Conclusion
Our findings suggest that the IFN-g-874 genotype may be a greater risk factor in the causation of AA, whereas the TNF-a-308 genotype has a protective role in the North Indian population.

Keywords: Aplastic anemia, TNF-a-308, IFN-g-874, Polymorphism, PCR-RFLP, ELISA

Fig 1.

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Figure 1.(A) TNF-α-308 (G/A) poly-morphism; GG: wild homozygous (107 bp), GA: heterozygous (107 bp, 87 bp, and 20 bp), AA: mutant (87 bp and 20 bp), L: molecular weight ladder (50 bp ladder). (B) IFN-γ-874 (A/T) polymorphism; AA: wild homozygous (176 bp), AT: heterozygous (176 bp, 148 bp, and 28 bp), TT: mutant (148 bp and 28 bp), L: molecular weight ladder (50bp ladder).
Blood Research 2020; 55: 193-199https://doi.org/10.5045/br.2020.2020009

Fig 2.

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Figure 2.Plasma levels of IFN-γ in patients vs. healthy controls (A). Plasma levels of TNF-α in patients vs. healthy controls (B).
Blood Research 2020; 55: 193-199https://doi.org/10.5045/br.2020.2020009

Table 1 . Genotyping information of TNF-α-308 and IFN-γ- 874 single nucleotide polymorphisms..

Gene SNP namePrimer sequence (5′–3′)Restriction enzymeRecognition sequenceWild type fragment lengthVariant (mutant) type fragment lengthHeterozygous type fragment length
TNF-α-308F:5′-AGGCAATAGGTTTTGAGGGCCAT-3′NCo15′-C|CATGG-3′107 bp (GG)87 bp and 20 bp (AA)107 bp, 87 bp, and 20 bp (GA)
R:5′-TCCTCCCTGCTCCGATTCCG-3′5′-GGTAC|C-3′
IFN-γ-874F:5′-GATTTTATTCTTACAACACAAAATCAAGAC-3′Hinf15′-G|ANTC-3′176 bp (AA)148 bp and 28 bp (TT)176 bp, 148 bp, and 28 bp (AT)
R:5′-GCAAAGCCACCCCACTATAA-3′5′-CTNA|G-3′

Abbreviation: bp, base pair..

Table 2 . Demographic details of patients with acquired aplastic anemia and healthy control subjects..

CharacteristicsPatients (N=120)Controls (N=120)
Age, mean yr±SD29.13±16.427.92±8.9
Sex
Male (%)83 (69.2)62 (51.7)
Female (%)37 (30.8)58 (48.3)
Patients classification on the basis disease severity
Severity
Severe (%)53 (44.2)0 (0)
Non-severe (%)56 (46.7)0 (0)
Very severe (%)11 (9.1)0 (0)
Patients categorization on the basis of response to immunosuppressive therapy
Response to immunosuppressive therapy
Responder (complete+partial) (%)63 (52.5)0 (0)
Non-responder (%)57 (47.5)0 (0)

Table 3 . Genotype and allele frequencies of the TNF-α-308 and IFN-γ-874 cytokine gene among patients with acquired aplastic anemia and healthy controls and their association with acquired aplastic anemia..

Gene polymorphismPatients (%), N=120Controls (%), N=120POR (95% CI)
TNF-α-308 (G/A) genotype
GG (wild)70 (58.3%)85 (70.8%)-Reference
AA (mutant)6 (5%)5 (6%)0.5460.68 (0.20–2.34)
GA (hetero)44 (36.7%)30 (36)0.043a)0.56 (0.32–0.98)
Allele frequency
G184 (0.77)200 (0.83)-Reference
A56 (0.23)40 (0.17)0.0670.65 (0.41–1.03)
Dominant70 (58.3%)85 (70.8%)-Reference
GG vs. GA+AA50 (41.7%)35 (29.2%)0.042a)0.57 (0.33–0.98)
Over-dominant76 (63.3%)90 (75%)-Reference
GA vs. GG+AA44 (36.7%)30 (25%)0.0500.57 (0.33–1.00)
Recessive114 (95%)115 (95.8%)-Reference
AA vs. GG+GA6 (5%)5 (4.2%)0.7570.82 (0.24–2.78)
IFN-γ-874 (A/T) genotype
AA (wild)66 (55.0%)55 (45.83%)-Reference
TT (mutant)20 (16.67%)15 (12.5%)0.7850.90 (0.42–1.92)
AT (hetero)34 (28.33%)50 (41.67%)0.047a)1.76 (1.00–3.10)
Allele frequency
A166 (0.70)160 (0.67)-Reference
T74 (0.30)80 (0.33)0.5571.12 (0.76–1.64)
Dominant66 (55.0%)55 (45.8%)-Reference
AA vs. AT+TT54 (45.0%)65 (54.2%)0.1551.44 (0.86–2.40)
Over-dominant86 (71.6%)70 (58.3%)-Reference
AT vs. AA+TT34 (28.4%)50 (41.7%)0.030a)1.80 (1.05–3.09)
Recessive100 (83.3%)105 (87.5%)-Reference
TT vs. AA+AT20 (16.7%)15 (12.5)0.3600.71 (0.34–1.47)

a)Statistically significant susceptible genotype..

Abbreviations: CI, confidence interval; OR, odds ratio..

Table 4 . Genotype distribution of the TNF-α-308 and IFN-γ-874 gene polymorphisms among patients with non-severe, severe, and very severe aplastic anemia vs. controls..

GenotypeSevere (N=53)Non-severe (N=56)Very-severe (N=11)Control (N=120)Severe patients and controlsNon-severe patients and controlsVery-severe and controls
POR (95% CI)POR (95% CI)POR (95% CI)
TNF-α-308 (G/A)
GG (wild)30 (56.60%)36 (64.28%)4 (36.36%)85 (70.83%)-Reference-Reference-Reference
AA (mutant)3 (5.66%)2 (3.57%)1 (9.09%)5 (4.16%)0.4410.58 (0.13–2.61)0.9991.05 (0.19–5.71)0.2820.23 (0.02–2.51)
GA (hetero)20 (37.73%)18 (32.14%)6 (54.54%)30 (25.0%)0.0730.52 (0.26–1.06)0.3300.70 (0.34–1.42)0.0320.23 (0.06–0.89)
Dominant30 (56.60%)36 (64.28%)4 (36.36%)85 (70.83%)-Reference-Reference-Reference
GG vs. GA+AA23 (43.39%)20 (35.71%)7 (63.63%)35 (29.16%)0.0670.53 (0.27–1.05)0.3820.74 (0.37–1.45)0.0370.23 (0.06–0.85)
Over-dominant33 (62.26%)38 (67.85%)5 (45.45%)90 (75.0%)-Reference-Reference-Reference
GG+AA vs. GA20 (37.73%)18 (32.14%)6 (54.54%)30 (25.0%)0.0880.55 (0.27–1.09)0.3210.70 (0.35–1.41)0.0350.27 (0.07–0.97)
Recessive50 (94.33%)54 (96.42%)10 (90.90%)115 (95.83%)-Reference-Reference-Reference
GG+GA vs. AA3 (5.66%)2 (3.57%)1 (9.09%)5 (4.16%)0.7010.72 (0.16–3.15)0.9991.17 (0.22–6.24)0.4150.43 (0.04–4.09)
IFN-γ-874 (A/T)
AA (wild)27 (50.94%)34 (60.71%)5 (45.45%)55 (45.83%)-Reference-Reference-Reference
TT (mutant)10 (18.86%)7 (12.5%)3 (27.27%)15 (12.5%)0.5150.73 (0.29–1.85)0.5781.32 (0.49–3.58)0.3760.45 (0.09–2.12)
AT (hetero)16 (30.18%)15 (26.78%)3 (27.27%)50 (41.67%)0.2471.53 (0.74–3.17)0.046a)2.06 (1.00–4.22)0.7211.51 (0.34–6.67)
Dominant27 (50.94%)34 (60.71%)5 (45.45%)55 (45.83%)-Reference-Reference-Reference
AA vs. AT+TT26 (49.05%)22 (39.28%)6 (54.54%)65 (54.16%)0.5341.22 (0.64–2.34)0.0651.82 (0.95–3.48)0.9800.98 (0.28–3.40)
Over-dominant37 (69.81%)41 (73.21%)8 (72.72%)70 (58.33%)-Reference-Reference-Reference
AT vs. AA+TT16 (30.18%)15 (26.78%)3 (27.27%)50 (41.66%)0.1511.65 (0.82–3.29)0.0561.95 (0.97–3.90)0.5231.90 (0.48–7.54)
Recessive43 (81.13%)49 (87.5%)8 (72.72%)105 (87.5%)-Reference-Reference-Reference
TT vs. AA+AT10 (18.86%)7 (12.5%)3 (27.27%)15 (12.5%)0.2720.61 (0.25–1.47)0.9991.00 (0.38–2.61)0.1760.38 (0.09–1.59)

a)Statistically significant susceptible genotype..

Abbreviations: CI, confidence interval; OR, odds ratio..

Table 5 . TNF-α-308 and IFN-γ-874 genotype distribution in responders (complete+partial responders) and non-responders..

Gene polymorphismComplete+partial responder (N=63)Non-responder (N=57)POR (95% CI)
TNF-α-308 genotype
GG (wild)35 (55.6%)35 (61.4%)-Reference
AA (mutant)4 (6.3%)2 (3.5%)0.6750.50 (0.08–2.91)
GA (hetero)24 (38.0%)20 (35.0%)0.6360.83 (0.39–1.77)
Dominant35 (55.6%)35 (61.4%)-Reference
GG vs. GA+AA28 (44.5%)22 (38.6%)0.5160.78 (0.37–1.62)
Over-dominant39 (61.9%)37 (64.9%)-Reference
GG+AA vs. GA24 (38.0%)20 (35.0%)0.7320.87 (0.41–1.85)
Recessive59 (93.6%)55 (96.4%)-Reference
GG+GA vs. AA4 (6.34%)2 (3.5%)0.6810.53 (0.09–3.04)
IFN-γ-874 Genotype
AA (wild)31 (49.2%)35 (61.4%)-Reference
AT (hetero)20 (31.7%)14 (24.5%)0.2610.62 (0.26–1.43)
TT (mutant)12 (19.1%)8 (14.1%)0.3070.59 (0.21–1.63)
Dominant31 (49.2%)35 (61.4%)-Reference
AA vs. AT+TT32 (50.8%)22 (38.6%)0.1790.60 (0.29–1.26)
Over-dominant43 (68.2%)43 (75.4%)-Reference
AA+TT vs. AT20 (31.8%)14 (24.6%)0.3830.70 (0.31–1.56)
Recessive51 (80.9%)49 (85.9%)-Reference
AA+AT vs. TT12 (19.1%)8 (14.1%)0.4610.69 (0.26–1.84)

Abbreviations: CI, confidence interval; OR, odds ratio..
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