The polymorphic landscape analysis of GATA1 exons uncovered the genetic variants associated with higher thrombocytopenia in dengue patients

Abstract

The current study elucidated an association between gene variants and thrombocytopenia through the investigation of the exonic polymorphic landscape of hematopoietic transcription factor-GATA1 gene in dengue patients. A total of 115 unrelated dengue patients with dengue fever (DF) (N = 91) and dengue hemorrhagic fever (DHF) (N = 24) were included in the study. All dengue patients were confirmed through detection of NS1 antigen, IgM, and IgG antibodies against the dengue virus. Polymerase chain reaction using specific primers amplified the exonic regions of GATA1 while Sanger sequencing and chromatogram analyses facilitated the identification of variants. Variants G>A (at chX: 48792009) and C>A (at chX: 4879118) had higher frequency out of 13 variants identified (3 annotated and 10 newly recognized). Patients carrying either nonsynonymous or synonymous variants had significantly lower mean values of platelets compared to those harboring the reference nucleotides (NC_000023.11). Further analyses revealed that the change in amino acid residue leads to the altered three-dimensional structure followed by interaction with neighboring residues. Increased stability of the protein due to substitution of serine by asparagine (S129N at chX: 48792009) may cause increased rigidity followed by reduced structural flexibility which may ultimately disturb the dimerization (an important prerequisite for GATA1 to perform its biological activity) process of the GATA1 protein. This, in turn, may affect the function of GATA1 followed by impaired production of mature platelets which may be reflected by the lower platelet counts in individuals with such variation. In summary, we have identified new variants within the GATA1 gene which were found to be clinically relevant to the outcome of dengue patients and thus, have the potential as candidate biomarkers for the determination of severity and prognosis of thrombocytopenia caused by dengue virus. However, further validation of this study in a large number of dengue patients is warranted. Trial Registration: number SLCTR/2019/037.

Fig 1. Graphical representation of the methodology undertaken during the study.

Fig 2. Agarose gel electrophoresis of the amplicons generated from PCR using different primer sets.

Primer set 1, 2, 3 and 4 covered the entire coding region of GATA1. For proper estimation of the size of the DNA bands, 100bp ladder DNA was used. S1 = Sample 1; S2 = Sample 2; NC = Negative Control.

Fig 3. Comparative analyses of the platelet counts.

(A) Between different groups of dengue patients with respect to variants recognized within the exonic regions of GATA1 gene with respect to nonsynonymous mutations and wild genotype (those harboring exactly the same sequence as the reference nucleotides). (B) Platelet counts of patients with nonsynonymous and wild type variants in GATA1 gene were stratified according to DF and DHF. Data has been presented as mean±SEM and p<0.05 was considered as the level of significance. Statistical analyses revealed that neither of the groups had significant differences.

Fig 4. Structure analyses of ab initio GATA1 modelled protein.

(A) Predicted 3D structure of the protein (B) Ramachandran plot of the structure (C) Z-score plot from PROSA (D) Residual error value plot from ERRAT2.

Fig 5. Surface structure of the modelled GATA1 protein with structural domains.

N-terminal activation domain, AD (red color), N-terminal Zinc finger domain, N-ZF (orange color) and C terminal Zinc finger domain, C-ZF (purple color).

Fig 6. Evaluation of the effect of nonsynonymous mutations on the structure and interactions between side chain and neighboring residues.

Among non-synonymous mutations, P21H (A) and S26T (B) mutations were found in the AD domain of the protein while Q262H (F) was within the C-ZF domain. Other mutations including S91L (C), G99S (D), S129N (E) and H289D (G) were found in regions outside the functional domains of GATA1 protein.