Association of hypercoagulability in COVID-19

patients with serum homocysteine level, MTHFR

gene polymorphisms, and serum SCUBE-1 level

A research proposal by:

Ibraheem Kais Taha
 Introduction
• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
causing coronavirus disease 2019 (COVID-19) has rapidly evolved
from an epidemic outbreak in Wuhan, China into a pandemic [1].

• Globally, as of 15 November 2021, there have been 253,163,330 confirmed

cases of COVID-19, including 5,098,174 deaths, reported to WHO [2].

• Signs and symptoms include: fever, cough, tiredness, loss of taste or smell,
SOB, muscle aches, sore throat, runny nose, headache, chest pain,
conjunctivitis, nausea, vomiting, diarrhea and rash[3].

• The lungs are the main organs involved, but systemic disease with wide
range of clinical manifestations may also develop, including multiple organ
failure, heart problems, acute kidney injury and additional viral and bacterial
infection. One of the major systems affected is the CVS [3,4]. Many patients
were found to have coagulation markers abnormalities [5].
• Hypercoagulability or thrombophilia is the increased tendency of blood to
thrombose. It describes the pathologic state of exaggerated coagulation or
coagulation in the absence of bleeding (e.g. venous thromboses, such as
deep venous thrombosis (DVT) and pulmonary embolism (PE)).

• Homocysteine (Hcy) is a non-proteinogenic amino acid that is formed in the

human body in methionine metabolism. Although not forming proteins,
homocysteine participates in major processes such as transmethylation,
cysteine (Cys) formation, transsulfuration, etc. In the transmethylation
process, homocysteine is an intermediate that allows the formation of
compounds with a major metabolic role such as adrenaline, lecithin, creatine,
etc. Cysteine formation, via homocysteine, is a very important process
because Cys is a vital amino acid to stabilize the spatial conformations of
proteins, to form the most important antioxidant agent in the body named
glutathione, or to detoxify harmful compounds.

• Elevated homocysteine level may result from deficiency or impaired function

of enzymes and cofactors in these pathways. Plasma homocysteine level is
influenced by many factors, genetic as well as environmental. Mild
hyperhomocysteinemia is quite common.
• The role of homocysteine in venous thrombosis has been studied less
extensively than its role in arterial diseases and nowadays it seems quite
controversial [6].

• Homocysteine assessment has been proposed as a potential predictive

biomarker for the severity of COVID-19 infection [7].

• Gene polymorphisms involved in homocysteine-methionine pathway result in

hyperhomocysteinemia.

• Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate and

homocysteine metabolism.

• The two known functional polymorphisms of MTHFR gene, 677C>T and

1298A>C have been implicated in a variety of multifactorial diseases: cardio-
cerebrovascular and neurodegenerative disorders, autoimmune diseases, birth
defects, diabetes, neuropsychiatric disorders, cancer and renal disease [8].
• SCUBE-1 (Signal peptide, CUB domain and EGF-like domain containing
protein-1)

• Etymology:
 Signal peptide:  is a short peptide (usually 16-30 amino acids long) present
at the N-terminus (or occasionally C-terminus]) of most newly
synthesized proteins that are destined toward the secretory pathway.
 CUB domain (for complement C1r/C1s, uEGF (urinary epidermal growth factor),
BMP1 (bone morphogenetic protein 1)): is a structural motif of approximately 110
residues found almost exclusively in extracellular and plasma membrane-
associated proteins, many of which are developmentally regulated. These
proteins are involved in a diverse range of functions, including complement
activation, developmental patterning, tissue repair, axon guidance and
angiogenesis, cell signaling, fertilization, haemostasis, inflammation,
neurotransmission, receptor-mediated endocytosis, and tumour
suppression.
 EGF-like domain: The EGF-like domain is an evolutionary
conserved protein domain, which derives its name from the EGF where it
was first described. It comprises about 30 to 40 amino-acid residues
including six cysteine residues which have been shown to be involved in
disulfide bonds.. EGF-like domains frequently occur in numerous tandem
copies in proteins: these repeats typically fold together to form a single,
linear solenoid  domain block as a functional unit.
• 3 Family members (SCUBE-1, SCUBE-2, and SCUBE-3) have an amino-
terminal signal peptide, nine copies of EGF-like repeats and a CUB domain at
the carboxyl terminus.

• SCUBE-1 first to be discovered in the year 2000.

• SCUBE1 has been shown to be elevated in patients with acute coronary

syndrome and ischemic stroke [9]. Higher SCUBE1 levels have been
demonstrated in an experimental model of acute ischemic stroke and acute
mesenteric ischemia [10,11]. This leads to plasma SCUBE1 being considered a
potential biomarker of platelet activation in acute thrombotic disease [12].
Aims of study:
• The association between serum homocysteine
levels and hypercoagulability in COVID-19 patient.

• The association between serum SCUBE-1 levels

and hypercoagulability in COVID-19 patient.

• The role of MTHFR gene polymorphism in serum

levels of homocysteine and SECUBE-1 as well as
in hypercoagulability in COVID-19 patients.
Materials and methods:
Study design
• Cross-sectional study.

• 120 patients with SARS-CoV-2 who will be admitted and

treated at Al-Imamain Al-Kadhimain Medical City, and
were diagnosed (by a consultant specialist in medicine)
after a +ve nasopharyngeal swab examination of SARS-
CoV-2 by RT-PCR, or –ve one with typical signs and
symptoms, typical findings on a CT-scan, and with no
other explanation of the symptoms (e.g., bacterial
infection)

• Patients haven’t received vaccination yet.

Inclusion criteria:
• All adult patients who are confirmed with SARS-CoV-2
infection.

Exclusion criteria:
• Patients who refuse to participate.
• Age < 18 years.
• Patients having anticoagulant treatment started within
48 hours before sampling.
Data Collection

• Demographic data (age, sex, BMI, residence, smoking and

comorbidities) – collected through direct interview.

• Laboratory data (CBC, s.ferritin, CRP, D-dimer, ESR and LDH) –

collected from patients’ records.

Methods

• This work will be done at the Medical Research Unit,

• 5 ml of blood collected from each patient at admission in order to avoid

the possible effects of treatment. 3ml in plane tube to obtain sera for
ELISA, and 2 ml in EDTA tube for molecular analysis.
• Homocysteine and SCUBE-1:

A ready commercial ELISA kits will be used for determination of

Homocysteine and SCUBE-1 levels according to the manufacturer’s
instructions.

• Molecular assay:

Gene amplification and genotyping

 DNA will be extracted from blood samples using a ready kit
(gSYNCTM DNA Mini Kit Whole Blood Protocol/Geneaid/Korea)
according to the manufacturer’s instructions.
 The extracted DNA from blood samples was used in a
polymerase chain reaction (PCR) for amplification of (MTHFR)
gene fragment corresponding to the C677T and A1298C
polymorphisms.
 Amplification Refractory Mutation System-PCR (ARMS-PCR) will be
performed using three primers for each mutation, one forward primer
and two reverse primers specific for the wild type and mutant alleles
that are shown in Table 1. The PCR products will be electrophoresed
on 2% agarose gel and stained with ethidium bromide to determine
the genotyping of each polymorphism.

Table 1: Primer for screening of MTHFR mutations by ARMS-PCR

Statistical Analysis
• All statistical analyses will be performed using SPSS software.
• Patients will be categorized into two groups: with and without
hypercoagulability according to serum levels of D-dimer.
• Hypercoagulability is defined in this study as >1.5 times increase in D-
dimer beyond the normal limit (normal limit is <= 500 ng/ml).
• The association of different demographic and clinical characteristics
with hypercoagulability will be calculated using Student t-test or Chi
square test.
• For genetic analysis, logistic regression test will be used. From this
test, the odds ratio and its corresponding 95%CI will be calculated. A
p-value of <0.05 will be considered significant.
References
1. Zhu N, Zhang D, Wang W, et al. A novel coronavirus from patients with
pneumonia in China, 2019. N Engl J Med. 2020;382(8):727–733.
doi:10.1056/NEJMoa2001017.
2. WHO coronavirus (COVID-19) dashboard. (n.d.-b). World Health
Organization (WHO). Retrieved November 15, 2021, from https://
covid19.who.int/
3. Coronavirus disease 2019 (COVID-19) - symptoms and causes. (n.d.).
Mayo Clinic. Retrieved November 10, 2021, from https://
www.mayoclinic.org/diseases-conditions/coronavirus/symptoms-cause
s/syc-20479963
4. Farshidfar, F., Koleini, N., & Ardehali, H. (2021). Cardiovascular
complications of COVID-19. JCI insight, 6(13), e148980.
https://doi.org/10.1172/jci.insight.148980.
5. Hirmerová, J. (2013). Homocysteine and venous thromboembolism- Is
there any link? Cor Vasa, 55(3), e248-258.
6. Hirmerová, J. (2013). Homocysteine and venous thromboembolism- Is
there any link? Cor Vasa, 55(3), e248-258.
7. Ponti, G., Pastorino, L., Manfredini, M., Ozben, T., Oliva, G., Kaleci, S.,
Iannella, R., & Tomasi, A. (2021). COVID-19 spreading across world
correlates with C677T allele of the methylenetetrahydrofolate reductase
(MTHFR) gene prevalence. Journal of clinical laboratory analysis, 35(7),
e23798. https://doi.org/10.1002/jcla.23798.
8. Carlotta Pia Cristalli, Chiara Zannini, Giorgia Comai, Olga Baraldi, Vania
Cuna, Maria Cappuccilli, Vilma Mantovani, Niccolò Natali, Giuseppe
Cianciolo and Gaetano La Manna. (2017). Methylenetetrahydrofolate
reductase, MTHFR, polymorphisms and predisposition to different
multifactorial disorders. Genes &amp; Genomics, 39(7), 689-699.
9. Dai, D. F., Thajeb, P., Tu, C. F., Chiang, F. T., Chen, C. H., Yang, R. B., &
Chen, J. J. (2008). Plasma concentration of SCUBE1, a novel platelet
protein, is elevated in patients with acute coronary syndrome and
ischemic stroke. Journal of the American College of Cardiology, 51(22),
2173–2180. https://doi.org/10.1016/j.jacc.2008.01.060.
10. Turkmen S, Eryigit U, Karaca Y, Mentese A, Sumer UA, Yulug E, et al.
Diagnostic value of plasma signal peptide-Cub-Egf domain-containing
protein-1(SCUBE-1) in an experimental model of acute ischemic
stroke. Am J Emerg Med. 2015;33:262–265.
11. Aköz, A., Türkdoğan, K. A., Kahraman Çetin, N., Kum, S., Duman, A.,
Türe, M., & Demirkıran, A. E. (2018). Predicting critical duration and
reversibility of damage in acute mesenteric ischemia: An experimental
study. Ulusal travma ve acil cerrahi dergisi = Turkish journal of trauma &
emergency surgery : TJTES, 24(6), 507–513.
https://doi.org/10.5505/tjtes.2018.69710.
12. Akdoğan, E., Ayaz, T., Kırbaş, A., & Rakıcı, H. (2019). Is SCUBE1 helpful
to predict the arterial thrombotic risk in patients with multiple myeloma: a
preliminary study. Hippokratia, 23(1), 21–24.