RESUMO
In our previous studies, we have demonstrated the association of certain variants of the thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), and thyroglobulin (TG) genes with congenital hypothyroidism. Herein, we explored the mechanistic basis for this association using different in silico tools. The mRNA 3'-untranslated region (3'-UTR) plays key roles in gene expression at the post-transcriptional level. In TSHR variants (rs2268477, rs7144481, and rs17630128), the binding affinity of microRNAs (miRs) (hsa-miR-154-5p, hsa-miR-376a-2-5p, hsa-miR-3935, hsa-miR-4280, and hsa-miR-6858-3p) to the 3'-UTR is disrupted, affecting post-transcriptional gene regulation. TPO and TG are the two key proteins necessary for the biosynthesis of thyroid hormones in the presence of iodide and H2O2. Reduced stability of these proteins leads to aberrant biosynthesis of thyroid hormones. Compared to the wild-type TPO protein, the p.S398T variant was found to exhibit less stability and significant rearrangements of intra-atomic bonds affecting the stoichiometry and substrate binding (binding energies, ΔG of wild-type vs. mutant: ‒15 vs. ‒13.8 kcal/mol; and dissociation constant, Kd of wild-type vs. mutant: 7.2E-12 vs. 7.0E-11 M). The missense mutations p.G653D and p.R1999W on the TG protein showed altered ΔG (0.24 kcal/mol and 0.79 kcal/mol, respectively). In conclusion, an in silico analysis of TSHR genetic variants in the 3'-UTR showed that they alter the binding affinities of different miRs. The TPO protein structure and mutant protein complex (p.S398T) are less stable, with potentially deleterious effects. A structural and energy analysis showed that TG mutations (p.G653D and p.R1999W) reduce the stability of the TG protein and affect its structure-functional relationship.
RESUMO
Blood collection in heparin tubes for cytogenetic, and ethylenediaminetetraacetic acid [EDTA] tubes for molecular genetics applications respectively, are routine practices everywhere. If blood samples are required for cytogenetics as well as DNA work, two samples from each animal are usually collected, which leads to wastage of time and money. The present study tried to explore the possibilities of collecting a single blood sample in a heparinised tube for use in both applications. Two blood samples were collected from the same animals; one in a heparin tube and the other in an EDTA tube. DNA was extracted and stored at the same temperature and for the same durations. Comparative studies revealed that the DNA samples extracted from blood using these two different coagulants give more or less the same quality of results especially for polymerase chain reaction [PCR] based applications in cattle. The purpose of the present study was to establish the possibility of using heparin blood for chromosomal studies as well as for molecular biology. Such a practice will obviously save time and money in collecting samples in duplicate