In Silico Analysis of Post-COVID-19 Condition (PCC) Associated SNP rs9367106 Predicts the Molecular Basis of Abnormalities in the Lungs and Brain Functions

Abstract

Long- or post-COVID-19 syndrome, which is also designated by WHO as Post COVID-19 Condition (PCC), is characterized by the persistent symptoms that remain after recovery from SARS-CoV-2 infection. A worldwide consortium of Long COVID-19 Host Genetics Initiative (Long COVID-19 HGI) identified an SNP rs9367106 (G>C; chr6:41,515,652, GRCh38, p = 1.76 × 10^-10, OR = 1.63, 95% CI: 1.40-1.89) that is associated with PCC. Unraveling the functional significance of this SNP is of prime importance to understanding the development of the PCC phenotypes and their therapy. Here, in Silico, I explored how the risk allele of this SNP alters the functional mechanisms and molecular pathways leading to the development of PCC phenotypes. Bioinformatic methods include physical interactions using HI-C and Chia-PET analysis, Transcription Factors (TFs) binding ability, RNA structure modeling, epigenetic, and pathway analysis. This SNP resides within two long RNA genes, LINC01276 and FOXP4-AS1, and is located at ~31 kb upstream of a transcription factor FOXP4. This DNA region, including this SNP, physically interacts with FOXP4-AS1 and FOXP4, implying that this regulatory SNP could alter the normal cellular function of FOXP4-AS1 and FOXP4. Furthermore, rs9367106 is in eQTL with the FOXP4 gene in lung tissue. rs9367106 carrying DNA sequences act as distant enhancers and bind with several transcription factors (TFs) including YY1, PPAR-α, IK-1, GR-α, and AP2αA. The G>C transition extensively modifies the RNA structure that may affect the TF bindings and enhancer functions to alter the interactions and functions of these RNA molecules. This SNP also includes an ALU/SINE sequence and alteration of which by the G>C transition may prevent IFIH1/MDA5 activation, leading to suppression of host innate immune responses. LINC01276 targets the MED20 gene that expresses mostly in brain tissues, associated with sleep disorders and basal ganglia abnormalities similar to some of the symptoms of PCC phenotypes. Taken together, G>C transition of rs9367601 may likely alter the function of all three genes to explain the molecular basis of developing the long-term symptomatic abnormalities in the lungs and brain observed after COVID-19 recovery.

Keywords: FOXP4; LINC01276; enhancer; polymorphism; post-COVID-19 condition; sleep disorder; transcription factor.

Figure 1

Physical interaction of rs9367106 carrying FOXP4-AS1 and LINC01276 with FOXP4. Chromosome 6 (x-axis, distance) is plotted against chromosome 6 (y-axis, distance) to visualize the interaction within the same chromosome. The interacting regions spanning LINC01276 and FOXP4 region are shown in (a) Adult lung cells (ENCODE) at 5 kb resolution by Hi-C (High-C, the lowest available resolution) and (b) in situ Mi-C (Micro-C, in h1ESC (human 1 Embryonic Cells) at 2 kb resolution. The square box in “Blue” shows interacting projections from the diagonal line pointing to the positions of both SNPs, FOXP4-AS1 and FOXP4 promoter. Both Hi-C and Mi-C show that the SNP (rs9367106) region with LINC01276 and FOXP4-AS1 physically interacts with the FOXP4 promoter and coding regions in two different experiments.

Figure 2

TADs and loops of the physically interacting SNP-FOXP4 region [17,18,19,20,21,22,23]. Analysis of various datasets shows that the rs9367106, FOXP4-AS1, and FOXP4 lie in both TADs and loops. The start and end point of the interacting and loop regions were shown for each study. The shortest distance includes loops of 83 kb spanning the SNP and FOXP4. The highest interaction, as “peaks”, encompasses just before the SNP and FOXP4 region, and both are included in the same interacting compartment.

Figure 3

Structural modeling of rs9367106 carrying the flanking sequence. Both pairwise base and centroid modeling show altered RNA structure in WT (G) and mutant RNA (C). MFE (Minimum Free Energy) indicates the stability of the RNA structure. (a) WT MFE secondary structure model (MFE, −72.90 kcal/mol), and (b) MT MFE (MFE, −52.50 kcal/mol), (c) WT centroid model (MFE, −69.00 kcal/mol), and (d) MT centroid model (MFE, −39.70 kcal/mol). MFEs in WT in both models are lower, indicating a more stable RNA structure than MT. Base pairing ability determines the stable folded structure. The color bar denotes the probability of being unpaired from lowest (blue, 0) to highest (red, 1). Increased unpaired residues (blue) in MT in both cases increased the instability. (e) Epigenetic analysis shows that rs0367106 (blue arrow) includes a SINE/ALU-Y sequence. Notably, the G>C transition disrupts the ALU-Y sequences, and disruption of the ALU-Y sequence prevents activation of innate immunity.

Figure 4

rs9367601 binds with TFs and changes the expression of target genes. (a) The 100 bp flanking sequences of both sides of the SNP are shown with rs9367106 G marked in red. (b) The transcription factor binds between 90 bp and 100 bp (G>C transition is in 99 bp) designated by the numbers within the box along the length are 1, 16, 17, 18, 19, and those are represented by the list as YY1, PPAR-α, IK-1, GR-α, and AP2αA, respectively. (c) The effect of G (WT) and risk allele (C) of rs9367106 on the expression of LINC01276, FOXP4-AS1, FOXP4 and MED20 for both SNPs. C represents those patients who have at least one C allele. (d) The normalized expression of LINC01276, FOXP4-AS1, FOXP4, and MED20 in healthy individuals and COVID-19 patients who developed PCC phenotypes after recovery from mild, moderate, severe, and critical infection. FOXP4 and MED20 expression are markedly increased synchronously in moderate and severe patients but not in critical patients.

Figure 5

The rs9367106 carrying flanking sequences act as an enhancer. (a) The target of rs9367106 carrying superenhancers that act on FOXP4 and associated genes in various tissues. Notably, although the superenhancer including rs9367106 interacts with FOXP4 in all tissues, but with different genes in different tissues. (b) Visual representation of superenhancer targets correlated with physically interacting genes. The superenhancer that originates within LINC01276 and interacts with FOXP4 . The purple color is the antisense FOXP4-AS1. (c) Mechanism of altered regulatory function by G>C transition of the SNP rs9367106. LINC01276 and FOXP4-AS1 RNA interact with FOXP4, and LINC01276 also interact with MED20 to develop PCC phenotypes in neuronal and lung tissues.

Figure 6

rs9367106-mediated modulation of TFs leading to molecular function. (a) Ranking of TFs mostly affected by rs9367106 with p-value and score of activation. (b) Scatter plot of these TFs (UMAP2) showing the highest activities they mediate through FOXP2 and WT1 (TP63) (both are highlighted in red) pathway (UMAP1). (c) heatmap shows (matched red box) the FOXP4 acts on the FOXP2-FOXP1 pathway, and MED20 mediates through PPARG and EPHA activation. (d) Molecular function of co-expressed TFs. The rs9367106 co-expressed genes in the Geneset are involved in various molecular functions, ranging from TFs activation to cell adhesion and DNA/protein binding activities.