Date of Award

2024

Degree Type

Thesis

Degree Name

MPhil in Biological and Biomedical Sciences

First Advisor

Dr. Najeeha T Iqbal

Second Advisor

Dr. Afsar Mian

Third Advisor

Dr. Irfan Hussain

Department

Biological and Biomedical Sciences

Abstract

This study investigates the Vitamin D Receptor (VDR) gene single nucleotide polymorphisms (SNP) Apa1 (rs7975232 in intron 8; C>A), and FokI (rs2228570 in exon 2; C>T) in COVID-19 patients in Pakistani population, and their possible association with COVID-19 susceptibility and severity. A case-control study was conducted using 196 SARS-CoV-2 infected cases and 81 healthy controls. Genotyping of VDR polymorphisms was performed using Tetra-ARMS PCR, and serum plasma levels of Vitamin D (VD), IgG, and IL-6 cytokine were analyzed using ELISA. Clinical records of patients were also retrieved for immune parameters including C-reactive protein (CRP), procalcitonin, D-Dimer, ferritin, and Lactate Dehydrogenase (LDH).
Among the biochemical parameters, serum plasma levels of VD, IL-6, and IgG were significantly higher in cases than control s (p< 0.05),and plasma IL-6, D-Dimer, Ferritin, and LDH levels were also statistically different among COVID-19 categories [Mild (n=37), Moderate (n=22), Severe (n=60), Critical(n=77)] (p< 0.05) (Chapter 2).
The study found that genotypes of ApaI C/A (62%) and FokI T/T (60%) were dominant in our study (Cases=196; Control=81). On the allele level, the C allele of ApaI (Major allele frequency: 0.6) and T allele of FokI (Major allele frequency: 0.8) were predominant. Also, the C allele of ApaI genotype was significantly associated with COVID-19 susceptibility [Chi-Square; p value: 0.0142; OR (95% CI): 1.471 (95% CI: 1.079 - 2.008)]. (Chapter 3).
However, we found no statistically significant association between VDR polymorphisms and plasma VD or IgG levels, but significant differences were noted in plasma LDH (p< 0.05) with FokI (T/C) genotype, of FokI showed significant difference in their mean plasma IL-6 levels (Chapter 3).
We found no significant association of any genotype with severity after removing confounding variables from the analysis (age and co-morbidities) (multinomial regression; p>0.01) (univariate analysis; p< 0.05 for age and co-morbidities). For predictive analysis using our data, we used Multinomial Regression Model and reported a higher probability of moderate COVID-19 for all ApaI genotypes and critical COVID-19 for all FokI genotypes (Chapter 3).
For the proof-of-concept objective, we examined the gene expression of VDR, Cathelicidin Anti[1]microbial Peptide (CAMP), and TNF-ɑ in THP-1 cells upon stimulation with VD. This stimulation led to upregulated levels of CAMP and VDR (ΔΔ Ct=3.24 and 2.75, respectively) (Chapter 4).
The rationale behind our research was to elucidate potential role of VDR gene SNP and VD signaling involved in immune response to SARS-CoV-2 infection, which could provide insights into the pathogenesis of the disease and inform potential therapeutic strategies. In conclusion, we reported C allele of ApaI to be associated with COVID-19 susceptibility. Biochemical parameters IL-6, IgG, and VD were higher in cases than controls, an association of LDH and ferritin with COVID-19 severity with ApaI and FoKI requires further confirmation in a larger cohort. The upregulation of CAMP and inhibition of TNF-a confirms an anti-inflammatory role of VD in immune regulation.

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1

Last Page

127

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