Potential impact on coagulopathy of gene variants of coagulation related proteins that interact with SARS-CoV-2

Thrombosis has been one of the complications of the Coronavirus disease of 2019 (COVID-19), often associated with poor prognosis. There is a well-recognized link between coagulation and inflammation, however, the extent of thrombotic events associated with COVID-19 warrants further investigation. Poly(A) Binding Protein Cytoplasmic 4 (PABPC4), Serine/Cysteine Proteinase Inhibitor Clade G Member 1 (SERPING1) and Vitamin K epOxide Reductase Complex subunit 1 (VKORC1), which are all proteins linked to coagulation, have been shown to interact with SARS proteins. We computationally examined the interaction of these with SARS-CoV-2 proteins and, in the case of VKORC1, we describe its binding to ORF7a in detail. We examined the occurrence of variants of each of these proteins across populations and interrogated their potential contribution to COVID-19 severity. Potential mechanisms by which some of these variants may contribute to disease are proposed. Some of these variants are prevalent in minority groups that are disproportionally affected by severe COVID-19. Therefore, we are proposing that further investigation around these variants may lead to better understanding of disease pathogenesis in minority groups and more informed therapeutic approaches.


Characterization of synonymous and missense variants of coagulation genes of
We found all synonymous (Supplementary Table S3) and missense (Supplementary Table S4 Polymorphism Database (dbSNP) [48] and characterized them in terms of (i) population pair usage (RSCPU) [52] [53], (vii) rare codon enrichment [54], (viii) and %MinMax codon usage 1 9 3 [55]. For nonsynonymous variants, we additionally used amino acid fraction matching in an 1 9 4 MSA, likelihood of the variant amino acid in an amino acid MSA, SIFT [56] [50], and Polyphen 1 9 5 [57] [50]. The fraction matching and MSA likelihood measures use sequence homology and may 1 9 6 imply selection against the variant. SIFT uses sequence homology as well as physical 1 9 7 properties of amino acids, while Polyphen uses multiple sequence and structural features to 1 9 8 predict the effect of amino acid substitutions. MFE of mRNA may affect stability of mRNA 1 9 9 transcripts, which will affect transcript abundance and translation. Codon and codon pair usage 2 0 0 have been shown to impact translation kinetics [58] [59], and their metrics may be useful in 2 0 1 assessing the impact of synonymous mutations on protein conformation and function [52]. For 2 0 2 all variants, we provide the corresponding identifier in dbSNP (rsid) [48].

0 3
We applied filters based on codon usage changes, mRNA MFE changes, and position 2 0 4 conservation to identify variants that were potentially impactful on protein expression or 2 0 5 conformation, which may affect interactions with SARS-CoV-2 proteins. Then, based on 2 0 6 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC ORF7a proteins, using 10,000 decoys. All plots form energy funnels. 2 3 7 Variants that may impact COVID-19 severity While over 700 variants from these genes were found in the studies, only 55 variants had a p-2 4 1 value less than 0.05; these are listed in Table 2 and Table 3. However, none of them are 2 4 2 significantly impactful when controlling for multiple hypothesis testing. Interestingly, only one of 2 4 3 these is a coding variant. We characterized the 55 variants in terms of miRNA binding, splicing, 2 4 4 mRNA minimum free energy, and sequence conservation, to understand how they may affect  Table 2 and Supplementary Table S1, and full data is given in Supplementary Table  2  4  9 1 6 In addition, known clinical consequences of these variants are summarized in Table 4  Patients carrying the T nucleotide depicted worsened progression for agerelated macular degeneration relative to WT (C) [69]; Chinese and Japanese carrying the T nucleotide lack an association with age-related macular degeneration, seen in Caucasian population studies, although was predicted as pathogenic [70]. NM_000062.2: c.1029+2110T>C European and Mediterranean patients carrying the C nucleotide did not depict a higher association with hereditary angioedema relative to WT [71]. NM_000062.2: c.1030-1975G>C The intronic polymorphism 1030 +1975G>C has no pathogenic influence on hereditary angioedema although predicted as pathogenic [71]. NM_000062.2: c.1030-20A>G Association of the G allele with age-related macular degeneration was predicted to decrease the variant splicing form SERPING1, decrease protein expression and potentially limit the regulation of the compliment system [72]. No association was observed for Chinese Han carrying the G nucleotide with age-related macular degeneration [73].
NM_000062.2: c.-2415G>A Chinese Han patients carrying the A nucleotide did not demonstrate an increased risk of polypoidal choroidal vasculopathy relative to WT (G) [74]. South Korean patients carrying the A nucleotide did not show association with an increased risk of leukemia relative to WT (G) [75]. Caucasians carrying the A nucleotide did not exhibit an increased risk of age-related macular degeneration relative to WT (G) [76].

NM_000062.2: c.52-696C>T
Patients carrying the T nucleotide did not display an increased risk for anterior uveitis relative to WT (C) [77]. Chinese Han carrying the T nucleotide did not display an increased risk for polypoidal choroidal vasculopathy relative to WT (C) [74]. Caucasians carrying the T nucleotide did not display an increased risk for age-related macular degeneration relative to WT (C) [76]. Chinese carrying the T nucleotide did not display an increased risk for diabetic retinopathy relative to WT (C) [78]. European and Mediterranean's carrying the T nucleotide did not display an increased risk for hereditary angioedema relative to WT (C) [71].

NM_000062.2: c.52-130C>T
Chinese carrying the T nucleotide did not display a different association with age-related macular degeneration relative to WT (C) [73]. Caucasians carrying 105 and is also made available for use under a CC0 license.
(which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.08.272328 doi: bioRxiv preprint the T nucleotide displayed worsened progression of age-related macular degeneration relative to WT (C) [79]. Patients carrying the T nucleotide depicted worsened symptoms of age-related macular degeneration relative to WT (C) [69]. Chinese carrying the T nucleotide responded poorer to anti-VEGF treatment relative to WT (C) [80].

NM_000062.2: c.685+659C>T
Caucasians carrying the A nucleotide failed to depict a greater association with AMD relative to WT (G) [76]. South Korean patients carrying the A nucleotide did not depict a greater association with leukemia relative to WT (G) [81]. Han Chinese carrying the A nucleotide did not depict a significantly greater association with age-related macular degeneration relative to WT (G) [74].

NM_000062.2: c.685+1100C>T
European and Mediterranean patients carrying the T nucleotide failed to show a greater association with hereditary angioedema relative to WT (C) [71].

NM_000062.2: c.1029+926G>T
European and Mediterranean patients carrying the T nucleotide failed to show a greater association with hereditary angioedema relative to WT (G) [71]. Chinese Han carrying the T nucleotide did not depict a greater association with polypoidal choroidal vasculopathy relative to WT (G) [74].

NM_000062.2: c.1029+1443G>C
European and Mediterranean patients carrying the C nucleotide failed to show a greater association with hereditary angioedema relative to WT (G) [71].

NM_000062.2: c.1029+2111G>A
European and Mediterranean patients carrying the A nucleotide failed to show a greater association with hereditary angioedema relative to WT (G) [71].

NM_000062.2: c.1438G>A
Patients carrying the A nucleotide did not depict a change in Tacrolimus dosage requirements for transplant operations relative to WT (G) [82]. Chinese Han patients carrying the A nucleotide did not show a higher association with age-related macular degeneration or polypoidal choroidal vasculopathy relative to WT (G) [73].  Prevalence of VKORC1 variants across populations 2 8 6 COVID-19 has spread to the entire world, affecting people with variable genetic and racial 2 8 7 backgrounds. Therefore, we explored ORF7a interactions with variants of VKORC1 found 2 8 8 across races. There are 160 missense VKORC1 variants in dbSNP and at least 27 which affect 2 8 9 warfarin sensitivity [84]. The most common variants are shown in Table 5. The locations of the 2 9 0 warfarin sensitive variation are shown in Fig 3. However, many warfarin resistance-causing 2 9 1 variants are not listed in dbSNP, and some do not include population frequency information. In    (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.08.272328 doi: bioRxiv preprint In the United States, COVID-19 has disproportionally affected African American populations.

0 2
We sought to investigate whether VKORC1 variants could be implicated in the susceptibility of 3 0 3 this population. We found that African and African American populations were much more likely 3 0 4 to have at least one synonymous variant that significantly changes codon and codon pair usage 3 0 5 in a relatively conserved position. Upon further investigation, we find that this is due to a single 3 0 6 3 4 1 VKORC1 and VKORC1L1 causes warfarin resistance in VKORC1 and warfarin sensitivity in

4 3
In addition, we examined similarities of ORF7a with VKORC1 interacting proteins. Two human 3 4 4 proteins are structurally similar to ORF7a and interact with VKORC1: CXADR, a Coxsackievirus 3 4 5 and Adenovirus receptor [91], and PCDH1, a Hantavirus receptor [92]. Both proteins are The alignment is largely confined to the beta sheets. presented with demographic data sufficient to determine race [112]. In addition, there is 4 1 9 increasing evidence that African-American populations are at higher risk of thrombotic events 4 2 0 with COVID-19, even when adjusting for common risk factors such as BMI, diabetes, 4 2 1 hypertension, and cardiovascular disease [113] [114]. This suggests a role for additional 4 2 2 molecular factors that can contribute to a predisposition to thrombosis in the presence of   factors. The binding of ORF7a and VKORC1 may also limit coagulation in the lungs by 4 4 5 preventing the reduction of vitamin K epoxide to active vitamin K. This interaction may be less 4 4 6 influential in patients with warfarin resistance due to increased production of VKORC1 protein or 4 4 7 modified VKORC1 conformation, resulting in increased coagulation and worse prognosis.