Insilico Analysis of Alpha-1 Antitrypsin Pathway in Ischemic Stroke Patients

doi:10.21203/rs.3.rs-4809512/v1

Ischemic stroke is one of the types of strokes related to the low oxygen supply to the brain cells. It is subdivided into cardioembolic stroke, large artery atherosclerosis stroke, small vessel occlusion and cryptogenic stroke. The stroke is symptomized by body weakness or paralysis on one side. Its development is correlated to the genetic variations of the SERPINA1 gene that produces variants such as Z-allele variant where glutamic acid is replaced with lysine, S-allele variant where glutamic is replaced by valine and the normal M-allele variant which leads to the synthesis of less amount of AAT coupled with its deficiency (AATD) and lowering of AAT activity against the neutrophil elastase that degrades elastin in the lung tissue reducing the permeation of oxygen to the blood stream. In this study, various computational tools such as Heartbio portal, genotype-tissue expression platforms have been employed to retrieve data that relates to the stroke. The pathways in which SERPINA1 participates are identified and the genes involved in them with a regulation activity relating to the stroke were analyzed. The identified genes can be applied as biomarkers to trace the potential development of the stroke in patients with cardiovascular diseases, diabetes, obesity and others. Additionally, the genes can be employed in the therapeutical treatments of the stroke patients.

SERPINA1

AAT

Cardioembolism

large artery atherosclerosis

Alpha-1 Antitrypsin Deficiency (AATD)

An ischemic stroke involves a blockage in blood flow in human brain. If blood flow doesn’t return quickly enough, an ischemic stroke will cause permanent brain damage or even cause death.

The blockage of blood flow is raised by a blood clot or a piece of fatty plaque which is broken off from the inside of a blood vessel. Ischemic stroke may have symptoms like drooping muscles on one side of the face, numbness on one side of the face or in one arm or leg, weakness or paralysis in one arm, leg, or side of the body, loss of sensation and abnormal sensations on one side of the body and also neck stiffness. Ischemic stroke can be caused by atherosclerosis, clotting disorders, atrial fibrillation, high cholesterol (hyperlipidemia), Type 2 diabetes and also microvascular ischemic disease. Medications and treatments used for ischemic stroke are thrombolytic drugs, endovascular mechanical thrombectomy, stroke rehabilitation [1].

1.1 Sub types of ischemic strokes

Ischemic stroke is subdivided into four categories as [3]:

Large artery atherosclerosis

This describes the progressive building of plaque in the largest artery of body (aorta). This occurs in the neck and the head region responsible for approximately 15% of ischemic stroke. Plaque is a sticky substance made up of cholesterol, fat, calcium and other material that builds up in the walls of arteries leading to the hardening of the blood vessel. It is caused by factors such as smoking, unhealthy diet, or genetic predisposition. Patients with high blood pressure, high cholesterol level, high fat content, obesity, diabetes and some forms of inflammation are subjective to large artery atherosclerosis while the factor of genetic predisposition describes the tendency of physical or mental cords or disorder to be able to be inherited by the next generation. This usually results from specific genetic variations that are often inherited from a parent. These genetic changes contribute to stroke development but do not cause it directly. This explains why some people with genetic predisposition can contract the stroke while others, even in same family may not.

Cardioembolism

This is a type of stroke that involves blood clot in the brain that occurs when the heart pumps rejected materials such as cell tissue debris, blood clot to the brain where they get stuck causing the blockage of blood vessels. This is approximately 30% of ischemic stroke. The main factors that can tribute to this kind of stroke are; fragments of calcifications that grow due to small deposits of calcium in the tissues, tumor tissue where by the tissue cells divide abnormally leading to blockage of the blood vessel or decrease in the diameter of blood vessels, tumors can be benign (not cancerous) or malignant (cancerous) and other factors such as fragment from vegetarian (septic or aseptic).

Small vessel occlusion

It refers to as subcortical stroke caused by the blockage or leaking of a small, deeply located branch of a large artery in the brain, initially due to high blood pressure. This results into movement and /or sensation difficulties in some sides of body. With eventual deprivation of oxygen supply and nutrients to the brain, the brain cells will die off.

Cryptogenic stroke: This refers to stroke caused by other factors besides cardioembolism, large artery atherosclerosis and small vessel occlusion. This kind of stroke is further divided into two: stroke with determined etiology and stroke with undetermined etiology. It is a stroke that is identified as cerebral stroke of obscure or unknown origin. The main cause of this stroke is not quantifiable neither is identifiable and it contributes to around 25–40% of all ischemic strokes. It can be symptomized by body weakness or paralysis on one side, dizziness, speaking difficulties and also confusion.

1.2 Alpha-1 antitrypsin role in ischemic stroke

Alpha-1 antitrypsin (AAT) is a protein belonging to the serpin superfamily encoded by the SERPINA1 gene in human, located on the long arm of chromosome 14 (14q32.1). The SERPINA1 gene provides instructions for preparing a protein called alpha-1 antitrypsin, which is a type of serine protease inhibitor (serpin). Alpha-1 antitrypsin is produced in the liver and then it is transported throughout the body through the blood [18].

AAT inhibits the enzyme neutrophil elastase which is released from white blood cells to fight infections providing protection to the lung tissue from neutrophil elastase. Neutrophil elastase, an enzyme, which can damage lung tissue if not properly controlled through its digestive activity of the elastin found in the alveolar region of the lungs. Regulation of neutrophil elastase activity is critical to main tissue homeostasis and preventing tissue damage [18].

1.3 Serpina1 variants

Mutations in the SERPINA1 gene [22] are associated with alpha-1 antitrypsin deficiency, which is a genetic disorder distinguished by low levels of alpha-1 antitrypsin in the blood. This protein also functions as a part of innate antiviral immunity. Any Defects in this gene can cause the deficiency of plasminogen activator inhibitor-1 (PAI-1 deficiency), and high concentrations of this gene product are connected with thrombophilia.

More than 100 variants of SERPINA gene have been connected with a condition, Alpha 1 Antitrypsin deficiency. AAT deficiency results in the development of lung diseases such as Emphysema, Chronic Obstructive Pulmonary Disease (COPD), Bronchiectasis and it can also predispose individual to liver diseases like Liver cirrhosis, Liver failure. Diagnosis of AAT deficiency involves blood tests to measure the levels of AAT and identifying any genetic mutations in the SERPINA1 gene [18].

Ischemic stroke occurs when the blood supply to the part of the brain is blocked or reduced, this will cut down the supply of oxygen and nutrients supply to the brain which will leads to the brain death, ischemic stroke is caused by the blocked artery in the brain where the blood vessel is narrowed or blocked which will reduce the blood flow.

Andrei & Balaji, (2023), found out that ischemic stroke is a sudden neurologic deficit that result from focal cerebral ischemia associated with permanent brain infarction, Common causes are atherothrombotic occlusion of large arteries; cerebral embolism, non-thrombotic occlusion of small, deep cerebral arteries. Normal variations in the circle of Willis and in the caliber of various collateral vessels, atherosclerosis, and other acquired arterial lesions can interfere with collateral flow, increasing the chance that blockage of one artery will cause brain ischemia.

Zhang, Ka et al. (2023) found out that ischemic stroke is a heterogeneous condition influenced by a combination of genetic and environmental factors. Ischemic stroke involves two critical stages including the disruption of cerebral blood supply, and the subsequent development of cerebral tissue hypoxia and necrosis. Cerebral small vessel disease (CSVD) contributes to approximately 20% of all strokes, including 25% of ischemic strokes Cerebral autosomal-dominant arteriopathy with stroke and ischemic leukoencephalopathy (CADASIL) is the first and most common inherited CSVD syndrome described in the literature. CADASIL arises from mutations in the NOTCH3 gene located on Chromosome 19p13, and is inherited in an autosomal-dominant manner.

Aleksandra Ekkerte et al. (2021), found out that Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is most common monogenic cause of ischemic stroke. It is caused by a pathogenic variant of NOTCH3 gene. Fabry’s disease is caused by GLA gene mutation resulting in lysosomal enzyme α galactosidase A deficiency. Retinal vasculopathy with cerebral leukodystrophy (RVCL) is an autosomal dominant disorder caused by mutation in the TREX1 gene. Other genes related to small-vessel disease resulting in lacunar ischemic and hemorrhagic stroke are COL4A1 and COL4A2; both are located on 13 chromosome (13q34 locus) and encode α1 and α2 chains of type IV collage. Two genes (PITX2 and ZFHX3), located in chromosomes 4q25 and 16q22 were identified to be significant risk factors of cardio embryonic CE stroke.

Ilaria Ferrarotti et al. (2024), found out that low alpha 1 antitrypsin level (AAT)serum level, resulting from variations in SERPINA1 causes alpha-1 antitrypsin deficiency (AATD), the most frequently found variants in AATD, with the Z variant present in most individuals diagnosed with AATD. However, there are many other less frequent variants known to contribute to lung and/or liver disease in AATD. This article identified a total of 7631 rare variants and 216 types of rare variant across 80 counties.

Gabriela Santos and Alice M Turner (2020) found that the alpha-1 antitrypsin (AAT) protein is encoded by the SERPINA1 gene on chromosome 14, and its main function is to inactivate neutrophil elastase (NE) upon insult to the lungs, such as smoking. During the absence of AAT, there is an imbalance of proteinases and anti-proteinases, which leads to the progression of emphysema and deterioration of lung function, resulting in chronic obstructive pulmonary disease (COPD). In some mutations, polymerization of AAT in alveolar macrophages and the presence of pro-inflammatory AAT polymers, previously reported to be obtained in bronchoalveolar lavage in Pi∗ZZ patients, contribute to the pathogenesis in AATD lungs.

Steven K. Feske (2021), found out that ischemic stroke is a cerebral infraction, with inadequate supply of blood to cerebral tissue. first there is a reversible loss of tissue function and, given enough time, infarction with loss of neurons and supportive structures. These events start with the loss of electrical function and progresses to disturbance of membrane function with calcium influx leading to calcium-dependent excitotoxicity, generation of reactive oxygen species.

3.1 Materials

The materials that were required for retrieval of datasets about the stroke included alpha-1 antitrypsin protein sequence as well as Serpina1 gene sequence.

3.2 Tools used

PDB: The Protein data Bank database is a world-wide database maintained by the consortium of global organizations that include RCSB (Research Collaboration for Structural Bioinformatics) in United states, PDBe in Europe, PDBj in Japan and Electron Microscopy Data Bank (EMDB) and others. The database provides global access to the user to observe and retrieve data concerning various proteins. The PDB provides three-dimensional data about proteins such as their three-dimensional structures for large biological molecules like proteins and nucleic acids. The information/data uploaded to the database is first verified authentic scientifically and mostly obtained from experiments using methods such as X-ray crystallography, Nuclear Magnetic Resonance (MNR) spectroscopy, cryo-electron microscopy and others. The database is accessed through its website http://www.rcsb.org/ and all data can be accessed by use of keywords or accession numbers through search or advanced search options.

GenBank Database: This is a DNA database that provides open access to annotated collection of all publicly available nucleotide sequences and their protein translations on the GenBank result page. The database is part of the International Nucleotide Sequence Database Collaboration that comprises of DNA Data Bank of Japan (DDBJ), European Nucleotide Archive (ENA), and GenBank itself. It is maintained by National Centre for Biotechnology Information (NCBI) at the National Library of Medicine under National Institute of Health in the United States. It provides all information about genes and their proteins which can be accessed through use of keywords especially gene names. GenBank is accessed through its website address http://www.ncbi.nlm.nih.gov/genbank .

GTEx: Genotype-Tissue Expression abbreviated for GTEx is a database project that utilizes state-of-the-art protocols to study and analyses the human gene expression and regulation. The database provides all kind of information about the gene expression in various tissues, sex of people, gender, and also age brackets to provide the activity track of the gene activity and any of the genetic variations can be studied and determined. It also exploits the use of WGS, WES and RNAseq methods for the obtaining of molecular data of the genes. The database can be accessed through its website address https://www.gtexportal.org/

UniProt: The Universal Protein database (UniProt) is a freely accessible protein database that avails its users with information about the protein. This information involves the protein sequence, function and others derived from the genome sequencing projects. It is maintained by the UniProt consortium comprising of European Bioinformatics Institute (EBI) in United Kingdom., Swiss Institute of Bioinformatics (SIB) in Switzerland, Protein Information Resource (PIR) in the United States. The database can also be accessed through https://www.uniprot.org/

PathCards: This database is found under the collection of Pathway Unification database. It is an integrated database of human biological pathways and their annotations. Human pathways are clustered into Super Paths based on the genes involved and their similarity in function/activity. It allows the user to view pathway networks connectivity within a superpath along with the genes involved. The pathways information is consolidated from around twelve (12) databases that are providing a comprehensive streamlined analysis of experiments. It can be accessed through the website address https://pathcards.genecards.org/ using the names of the genes inserted into the search option provided.

Heartbio Portal: This is a data collection of all cardiovascular disease genetics data that provides computational infrastructural platform for visualizing, analyzing and downloading publicly available cardiovascular disease datasets. The datasets comprise of gene expression, genetic association as well as ancestry information extracted from different organizations such as Ensembl, ClinVar, NHGRI-EBI GWAS catalog, OMIM, GTEx and others. The GWAS datasets are filtered to a p-value less than 0.05. The portal employs limma to determine genes that are expressed differently across experimental conditions and variants found in known protein product correlated with various phenotypes. All the available data can be accessed and downloaded freely for publishing without restrictions. The database is accessed through its website address https://www.heartbio.portal.com/ and results are retrieved with use of a keyword search.

3.3 Methods and protocol

UniProt

The website is accessed through its address and the name of the protein – Alpha-1 antitrypsin – was used as a query keyword in the search box. On the results page, the result card with A1AT_HUMAN of entry number P01009 and sequence length 418 amino acids was selected. On the next page, the gene responsible for the synthesis of alpha-1 antitrypsin protein was identified, observed and analyzed. The protein sequence was retrieved and stored in FASTA format for use in next steps.

PDB

This database was accessed also and the protein name was used as the search term and the structural data of entry 1HP7 derived from Homo sapiens using X-ray crystallography was selected, observed and analyzed.

GenBank

The data about the SERPINA1 gene was obtained from the database through use of the gene name in the search box and the information about the gene including its location in the Homo sapiens species was retrieved and analyzed.

GTEx

In the search box of the database, the gene name was inserted and the gene was selected from the result table. The gene expression in bulk and single tissue was observed and analyzed. Using the Expression PCA (Principal Component Analysis), the expression of the gene the liver was observed for the sample traits that included age bracket, autolysis score, Hardy scale, ischemic time and sex.

PathCards

On the website homepage, the gene name was also used in the Search PathCards box as the search term. On the result page, the pathways in which SERPINA1 is related, connected or present were noted and the genes in those pathways were used to obtain information in the Heartbio portal in the next steps.

Heartbio Portal

The datasets from this portal were retrieved through use of the genes’ names from the pathways in PathCards. On the result page, the results were obtained for the p-value set to less than 0.05 in all traits [17]. The genetic association datasets, differential expression of genes in relation the stroke and genetic variants were downloaded, observed and analyzed.

From the PathCards database, thirteen (13) pathways were identified to have involvement and participation of the serpina1 gene or its protein. These pathways included the following;

1) Embryonic and induced pluripotent stem cells and lineage markers pathway.

2) P73 transcription factor network pathway.

3) Nuclear receptors meta pathway.

4) FOXA1 transcription factor network pathway.

5) Vesicle mediated transport pathway.

6) Metabolism of proteins pathway.

7) Lung fibrosis pathway.

8) Response to elevated platelet cytosolic Ca pathway.

9) Transport to the golgi and subsequent modification pathway.

10) Diseases of hemostasis pathway.

11) Regulation of insulin-like growth factors (IGF) transport and uptake by insulin-like growth factors binding proteins (IGFBPs) pathway.

12) Innate immune system pathway that included the neutrophil degranulation pathway.

13) COPII-mediated vesicle transport pathway.

Out of these pathways, genes related to the stroke in terms of differential expression have been analyzed with help of Heartbio portal and around 1800 genes have been identified to have relation to stroke either pre- or post-stroke occurrence.

These genes in their respective pathways have been clustered into tables presenting the gene name, the kind of ischemic stroke to which it is involved/related, the genetic variants and the protein identification numbers or accession numbers that can avail complete information about the gene and its protein as well as the variants in existence.

Keywords words used in the tables below;

Cardioembolic
Large artery atherosclerosis
Small vessel – this means small vessel occlusion stroke
Others – these include those with determined etiology other than cardioembolic, large artery atherosclerosis or small vessel and those with undetermined etiology
Variants – in the gene
Protein ID – given in form of nucleotide data ID because the gene can be accessed in GenBank and its protein translation is provided on the GenBank file.
NA – for “Not Available” represents that the particular data about the gene is not available at the moment of writing.

Table 1

represents the genes that are involved in lung fibrosis pathway that are connected to the regulation of ischemic stroke.
Genetic Association and Differential expression with stroke
Gene	Regulation	Stroke subtype (phenotype)	Variants	Protein ID
Lung fibrosis pathway
SERPINA1	Up	Cardioembolic, Large artery atherosclerosis & others	intron variant	NM_001002235.2 NM_000295.4 NM_001002235.2
CCR2	Up	Others Cardioembolic, Large artery atherosclerosis	synonymous variant, Upstream gene variant	NM_001123396.1 NM_001123041.2 NM_001123041.2
CCR3	Down	cardioembolic	Intron variant	NM_178328.1
CEBPB	Up & down	Others	Downstream gene variant	NM_005194.3
FAM13A	Up & down	Cardioembolic, Small-vessel occlusion	Intron variant, downstream gene variant	NM_000589.3 NM_172348.2 NM_000589.3
FGF1	Up	NA	Missense	NM_000800.4
GREM1	Up	Cardioembolic, large artery atherosclerosis, & others	Intron & upstream variant	NM_013372.6 NM_001191322.1 NM_001191323.1
PDGFB	Down	Large artery atherosclerosis & others	Intron & upstream variant	NM_002608.3 NM_033016.3
PLAU	Up	Cardioembolic, large artery atherosclerosis, small vessel and others	Downstream, intron, splice and 3’ UTR variants	NM_002658.4 NM_001145031.2 NM_001319191.1
TGFB1	Up	Others	Intron variant	NM_000660.6
TIMP1	Up	NA	NA	NA

The genes such as FAM13A, CCR2, CCR3 and others in this pathway have participation roles in the repair of the lung tissue and any sort of dysregulation leads to the scarring of the alveolar region reducing the permeability of oxygen in to blood for supply.

Table 2

represents the embryonic and induced pluripotent stem cells and linear markers which included genes responsible for embryogenesis and development. Genes like CXCR4, LMO2, and others have advanced roles in strokes such as small vessel and large artery atherosclerosis.
Embryonic and induced pluripotent stem cells and linear marker pathway
Gene	regulation	Stroke subtype (phenotype)	Variants	Protein ID
ATF2	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Synonymous, intron, splice-region, & sequence feature variants	NM_005171.4 NM_001256090.1 NM_001256091.1 NM_001256092.1
CXCR4	Up	Small vessel	Synonymous variant	NM_001008540.1
FGF18	Up & down	Cardioembolic & large artery atherosclerosis	Intron variant	NM_003862.2
GATA2	Down	Small vessel, large artery atherosclerosis & others	5’ UTR, intron, missense, upstream variants	NM_001145661.1 NM_032638.4
HNF1B	Down	Small vessel, Cardioembolic, large artery atherosclerosis & others	3’ UTR, intron & upstream variants	NM_001165923.3 NM_000458.3 NM_001304286.1
ITGB1	Up	Small vessel, Cardioembolic, large artery atherosclerosis & others	5’ UTR, intron, downstream, & upstream gene variants	NM_133376.2 NM_033668.2 NM_002211.3
KDR	Up	Cardioembolic, large artery atherosclerosis & others	Intron, missense, & structural interaction variants	NM_002253.2
KLF5	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, intron & downstream gene variants	NM_001286818.1 NM_001730.4
LGR4	Down	Cardioembolic, large artery atherosclerosis & others	Intron variant	NM_018490.2
LMO2	Up	Large artery atherosclerosis & others	Synonymous, intron & upstream gene variant	NM_001142315.1 NM_005574.3 NM_001142316.1
LMO4	Up & down	Small vessel & others	Intron variant	NM_006769.3
LRIG1	Down	Cardioembolic, Large artery atherosclerosis & others	Intron & missense variants	NM_015541.2
ONECUT2	Up & down	Cardioembolic, large artery atherosclerosis & others	3’ UTR & intron variants	NM_004852.2
PAX4	Up	NA	Missense & synonymous	NM_006193.2
PROX1	Down	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, downstream, 3’ UTR & intron variant	NM_001270616.1 NM_002763.4
SLC2A2	Up	Cardioembolic, large artery atherosclerosis & others	Downstream, upstream, intron, synonymous & missense variants	NM_001278658.1 NM_000340.1
SOX9	Down	Others	Intron variant	NM_000346.3
TCF3	Down	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, downstream, synonymous, splice-region, intron, 3’ UTR & missense	NM_003200.3 NM_001136139.2
THPO	Up	Cardioembolic, small vessel & others	Upstream, downstream & intron variants	NM_001290003.1 NM_000460.3 NM_001177597.2 NM_001290026.1

Table 3

shows the genes having connected activities to the p73 gene. These activities involve cell cycle arrest, apoptosis and others as discussed further in findings and discussions.
p73 transcription factor pathway
Gene	regulation	Stroke subtype (phenotype)	Variants	Protein ID
ADA	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Downstream, intron, structural interaction, missense, 5’ UTR, upstream, noncoding transcript variant	NM_000022.3 NM_001322050.1 NM_001322051.1
BAK1	Down	Large artery atherosclerosis	Intron variant	NM_001188.3
BAX	Up	Cardioembolic, large artery atherosclerosis, small vessel	Intron, downstream, upstream gene variant	NM_001291428.1 NM_001291429.1 NM_001291430.1 NM_004324.3 NM_138761.3
BIN1	Down	NA	Missense & synonymous	NM_139343.2
BUB1	Up	Cardioembolic, small vessel & others	Intron, sequence feature, upstream, downstream, splice-region variants	NM_001278616.1 NM_004336.4 NM_001278617.1
CCNA2	Up	Large artery atherosclerosis	Intron & 5’ UTR variant	NM_001237.3
CDK2	Up	Small vessel	Downstream gene variant	NM_001798.4 NM_001290230.1
CDKN1A	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, intron, 5’ UTR, synonymous, missense, 3’ UTR, sequence feature & downstream gene variant	NM_078467.2 NM_000389.4 NM_001220777.1 NM_001291549.1
FBXO45	Up	Others	Intron variant	NM_00105573.1
GDF15	Up	NA	Missense, synonymous & conservative in-frame insertion variant	NM_004864.2
HSF1	Down	Large artery atherosclerosis	Intron variant	NM_005526.2
IL1RAP	Up	NA	Missense, synonymous, & splice region variants	NM_001167931.1 NM_001167928.1
IL4R	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	3’ UTR, downstream, upstream, splice region, sequence feature & intron	NM_000418.3 NM_001257406.1 NM_001257407.1
JAK1	Up	NA	Missense & synonymous	NM_001320923.1 NM_001321857.1
MAPK14	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, 3’ prime, intron, downstream & sequence feature variants	NM_139013.2 NM_139012.2 NM_001315.2
NSG1	Down	Small vessel, cardioembolic & others	Intron, upstream & downstream gene variants	NM_001040101.1 NM_001287763.1 NM_001287764.1
PEA15	Down	Large artery atherosclerosis, cardioembolic & others	Splice-region, intron, 3’ UTR, 5’ UTR, downstream & upstream gene variants	NM_001297576.1 NM_001297577.1 NM_001297578.1 NR_123724.1
PML	Up & down	NA	Missense variants	NM_033239.2 NM_033250.2 NM_033238.2 NM_033249.2
RAD51	Up	Large artery atherosclerosis & cardioembolic	Intron, upstream downstream, 3’ UTR & 5’ UTR variants	NM_001164269.1 NM_001164270.1 NM_002875.4 NM_133487.3
RB1	Up	Large artery atherosclerosis, cardioembolic & others	Intron variant	NM_000321.2
RELA	Down	Small vessel, large artery atherosclerosis & others	Downstream, intron & upstream variants	NM_001145138.1 NM_001243984.1 NM_021975.3
SFN	Up	NA	Synonymous variant	NM_006142.3
SP1	Up	Cardioembolic, large artery atherosclerosis & others	Downstream, intron & upstream variants	NM_138473.2 NM_001251825.1 NM_003109.1
TP63	Up	NA	Synonymous variant	NM_0061422.4 NM_001329964.1 NM_001114982.1 NM_001329146.1 NM_001329148.1 NM_001114980.1
TUBA1A	Up	Large artery atherosclerosis	Intron variant	NM_001270399.1 NM_001270400.1

Table 4

represents the nuclear receptors that shows relatedness to the stroke regulation in terms of their gene’s expression during pre- or post-stroke development.
Gene	regulation	Stroke subtype (phenotype)	Variants	Protein ID
ABCB1	Down	Large artery atherosclerosis, cardioembolic & others	Intron, 5’ UTR & 3’ UTR variants	NM_000927.4
ABCC3	Down	Cardioembolic, large artery atherosclerosis & small vessel	Upstream & intron variants	NM_003786.3 NM_001144070.1
ABCG8	Up	Large artery atherosclerosis, small vessel, cardioembolic & others	Upstream, intron, synonymous & missense variants	NM_022437.2
ACKR3	Down	Large artery atherosclerosis & cardioembolic	Intron & synonymous variants	NM_020311.2
ALAS1	Up	Cardioembolic	Upstream, intron & downstream gene variants	NM_001304444.1 NM_001304443.1 NM_000688.5 NM_199166.2
APOA2	Up	NA	Missense & synonymous	NM_001643.1
APOA5	Up	Cardioembolic, small vessel & others	Downstream & upstream gene variants	NM_001166598.1
ARL5B	Up	NA	Splice region & synonymous	NM-178815.3
ARNT	Up	Cardioembolic & large artery atherosclerosis	Upstream, downstream, intron, 3’ UTR variants	NM_001668.3 NM_001197325.1 NM_001286035.1 NM_001286036.1
BIRC2	Up	Cardioembolic, small vessel & large artery atherosclerosis	Intron, sequence feature, upstream, 5’ UTR, downstream gene variants	NM_001256163.1 NM_001166.4 NM_001256166.1
BLVRB	Down	NA	Missense & synonymous	NM_000713.2
CAP2	Down	Large artery atherosclerosis, small vessel & others	Intron variant	NM_006366.2
CBR3	Down	Cardioembolic	Structural interaction variant	NM_001236.3
CCL20	Down	Large artery atherosclerosis & cardioembolic	Upstream & downstream variant	NM_004591.2
CPT1A	Up	Cardioembolic, small vessel & others	Upstream, downstream, intron, missense, synonymous, splice region	NM_001876.3 NM_001031847.2
CPT2	Up	Small vessel & others	Intron & downstream	NM_000098.2
CYP1A2	Down	Others	Intron & synonymous	NM_000761.4
CYP1B1	Up	Others	Intron variant	NM_000104.3
CYP2C9	Up & down	Cardioembolic, large artery atherosclerosis, small vessel & others	Intron & sequence feature variants	NM_000771.3
CYP3A5	Down	Large artery atherosclerosis, small vessel & others	Intron, splice acceptor, downstream gene variants	NM_000777.4 NR_033807.2 NM_001291829.1 NM_001291830.1
DNAJB1	Up	Cardioembolic & large artery atherosclerosis	Upstream & 3’ UTR variant	NM_006145.2 NM_001300914.1
EPB41L4B	Up & down	Cardioembolic, large artery atherosclerosis, small vessel & others	Intron, missense, splice region, upstream,	NM_019114.4
EPHA3	Down	NA	Missense, synonymous & splice region variant	NM_005233.5
ETNK2	Down	Cardioembolic, large artery atherosclerosis, small vessel & others	Missense, intron, upstream & downstream gene variants	NM_018208.3 NM_001297760.1 NM_001297761.1 NM_001297762.1
FGD4	Up	NA	Missense & synonymous variants	NM_001304480.1 NM_001304481.1 NM_139241.3 NM_001304484.1 NM_001304483.1
FTH1	Up	NA	Missense & synonymous	NM_002032.2
GADD45B	Up	NA	Missense & intron variant	NM_015675.3
GCLM	Up	Cardioembolic, large artery atherosclerosis & others	Downstream, upstream & intron variants	NM_002061.3 NM_001308253.1
GPR153	Up	Others	Downstream, intron, missense & synonymous	NM_207370.2
GSR	Up	Small vessel, cardioembolic & others	Intron, upstream, 3’ UTR, synonymous & missense variants	NM_000637.3 NM_001195102.1 NM_001195103.1 NM_001195104.1
GSTP1	Down	Small vessel	Missense, synonymous & intron variants	NM_000852.3
GSTT2	Up	NA	NA	NA
IP6K3	Down	Cardioembolic, large artery atherosclerosis, small vessel & others	Downstream, intron, synonymous, 5’ UTR & upstream gene variants	NM_001142883.1 NM_054111.4
IRS2	Up	Cardioembolic, large artery atherosclerosis & others	Intron & 3’ UTR variants	NM_003749.2
KTN1	Up	NA	Missense, synonymous & splice region variants	NM_001079521.1 NM_001079522.1 NM_004986.3 NM_001271014.1
MAFF	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, downstream, intron, 5’ UTR & 3’ UTR variants	NM_001161572.1 NM_012323.3
MGST1	Up	Small vessel & others	Upstream, sequence feature, intron, downstream gene variants	NM_001260511.1 NM_145792.2 NM_001260512.1 NM_020300.4 NR_048545.1
MGST2	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, intron, downstream gene variants	NM_001204366.1 NM_001204368.1 NM_002413.4
MYOF	Up	NA	Missense, synonymous, splice region & frameshift	NM_013451.3 NM_133337.2
NAV3	Up	NA	Missense, synonymous & splice region variant	NM_001024383.1 NM_014903.5
NCOA2	Up	NA	Missense & synonymous variant	NM_001321703.1 NM_001321712.1 NM_001321713.1
NR1H3	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, downstream, intron, 5’ UTR & synonymous	NM_001251934.1 NM_001251935.1 NM_005693.3 NM_001130102.2
NR1H4	Up	NA	Missense, synonymous & splice region variant	NM_001206993.1 NM_001206977.1 NM_005123.3 NM_001206992.1
NR3C1	Up	NA	Missense & synonymous	NM_001024094.1
PLK2	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Downstream, intron, synonymous, 5’ UTR, 3’ UTR, sequence feature & splice region variants	NM_006622.3 NM_001252226.1
PMP2	Down	NA	Missense & synonymous	NM_002677.3
POLK	Up	Small vessel & large artery atherosclerosis	Intron variant	NM_016218.2
PRDX6	Up	Cardioembolic	Intron variant	NM_004905.2
PTGS2	Up		Missense & synonymous	NM_000963.3
SERPINB2	Up	Cardioembolic & small vessel	Missense, intron & downstream gene variant	NM_001143818.1 NM_002575.2
SLC2A11	Down	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, intron, missense, non-coding transcript exon, 3’ UTR, downstream, 5’ UTR & synonymous	NM_001282864.1 NR_104248.1 NM_001024938.3 NM_030807.4
SLC2A12	Up	Large artery atherosclerosis, small vessel & others	Intron variant	NM_145176.2
SLC2A2	Up	Cardioembolic, large artery atherosclerosis & others	Downstream, intron, missense, synonymous, upstream variants	NM_000340.1 NM_001278658.1
SLC2A3	Up	Large artery atherosclerosis	Intron, synonymous, sequence feature,	NM_006931.2
SLC2A9	Up	NA	Missense & synonymous	NM_020041.2 NM_001001290.1
SLC39A1	Up	Cardioembolic, large artery atherosclerosis & others	3’ UTR & upstream gene variant	NM_001271957.1 NM_001271959.1 NM_001271958.1 NM_001271961.1 NM_014437.4
SLC39A4	Up	NA	Missense, synonymous & splice region variants	NM_017767.2 NM_130849.3 NM_001280557.1
SLC39A6	Up & down	Cardioembolic, large artery atherosclerosis, small vessel & others	Downstream, upstream, intron, synonymous,	NM_012319.3 NM_001099406.1
SLC39A9	Up	Small vessel & large artery atherosclerosis	Upstream, intron & 3’ UTR variant	NM_018375.4 NM_001252151.1 NM_001252152.1 NM_001252148.1 NM_001252150.1
SLC5A1	Up	Cardioembolic & large artery atherosclerosis	Downstream, upstream, intron, synonymous, missense & 3’ UTR variants	NM_001256314.1 NM_000343.3
SLC5A8	Up	Cardioembolic, large artery atherosclerosis & small vessel	Intron variant	NM_145913.3
SLC5A9	Up	Cardioembolic, large artery atherosclerosis & others	Synonymous, intron, upstream & missense variant	NM_001135181.1 NM_001011547.2
SLC6A1	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Intron variant	NM_003042.3
SLC6A2	Up	Cardioembolic, large artery atherosclerosis & others	Upstream, downstream, 5’ UTR, missense, intron,	NM_001172504.1 NM_001172501.1 NM_001043.3 NM_001172502.1
SLC7A11	Up	Cardioembolic, large artery atherosclerosis & others	Intron & sequence feature variant	NM_014331.3
SNAI2	Up	NA	Intron variant	NM_003068.4
SP1	Up	Cardioembolic, large artery atherosclerosis & others	Upstream, downstream & intron variant	NM_138473.2 NM_001251825.1 NM_003109.1
SQSTM1	Up & down	Cardioembolic, large artery atherosclerosis & others	Upstream, downstream, 5’ UTR, intron, synonymous & sequence feature variant	NM_001142298.1 NM_003900.4
SRC	Down	Cardioembolic, large artery atherosclerosis, small vessel & others	Intron, 5’ UTR & upstream gene variants	NM_005417.4 NM_198291.2
SREBF1	Up	Cardioembolic, large artery atherosclerosis & others	5’ UTR, 3’ UTR, downstream, upstream & intron variant	NM_001005291.2 NM_001321096.2 NM_004176.4
SRGN	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Downstream, upstream, 3’ UTR, intron variants	NM_001321053.1 NM_001321054.1 NM_002727.3
TGFBR3	Up	NA	Synonymous, missense & splice region variants	NM_003243.4 NM_001195683.1
TNFAIP3	Up	Small vessel, large artery atherosclerosis & others	Downstream, upstream & intron variants	NM_001270507.1 NM_001270508.1 NM_006290.3
TSC22D3	Down	NA	NA	NA
TXNRD1	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Synonymous, upstream, intron, downstream, 3’ UTR variants	NM_001093771.2 NM_001261445.1 NM_001261446.1 NM_003330.3 NM_182729.2 NM_182742.2 NM_182743.2
VDR	Up	Large artery atherosclerosis, small vessel & others	Downstream & intron variants	NM_001017536.1 NM_001017535.1 NM_000376.2

Table 5

depicts the genes connected to the Forkhead box A1 (FOXA1) factor.
FOXA1 transcription factor pathway
Gene	regulation	Stroke subtype (phenotype)	Variants	Protein ID
AR	Down	NA	Missense & synonymous	NM_000044.3 NM_001011645.2 NM_00112707.2
NR2F2	Up & down	NA	Synonymous variant	NM_021005.3 NM_001145155.1
POU2F1	Up	NA	Missense & synonymous	NM_002697.3 NM_001198786.1 NM_001198783.1
PRDM15	Down	NA	Missense, synonymous & splice region variant	NM_022115.4 NM_00104042.2 NM_001282934.1

Table 6

illustrates the genes involve in the hemostasis diseases pathway and thus these genes can be used as markers to depict the possibility of ischemic stroke in patients with disorders such as hemophilia, factor V Leiden thrombophilia and others.
Disease of hemostasis pathway
Gene	regulation	Stroke subtype (phenotype)	Variants	Protein ID
C3AR1	Up	Cardioembolic & large artery atherosclerosis	Downstream gene variant	NM_001326477.1 NM_001326475.1
C8G	Up	NA	Missense, synonymous & splice region variant	NM_000606.2
CD46	Up	NA	Missense, synonymous & splice region variant	NM_172359.2 NM_153826.2 NM_172350.2 NM_172361.2
CFD	Up	Cardioembolic, small vessel & others	Upstream & 3’ UTR variant	NM_001317335.1 NM_001928.3
CR2	Down	Cardioembolic & small vessel	3’ UTR, upstream, downstream & intron variants	NM_001006658.2 NM_001877.4
F11	Up	Cardioembolic, large artery atherosclerosis & others	Intron & sequence feature variants	NM_000128.3
F12	Up	NA	Missense, synonymous & splice region variant	NM_000505.3
F5	Up	Cardioembolic, small vessel, large artery atherosclerosis & others	Intron, synonymous, missense & 3’ UTR variant	NM_000130.4
F8	Up	NA	Missense & synonymous variant	NM_000132.2 NM_019863.2
FGA	Up	Cardioembolic, large artery atherosclerosis & others	3’ UTR, missense, intron, downstream, 5’ UTR, upstream,	NM_000508.4
GP5	Down	Large artery atherosclerosis	3’ UTR variant	NM_004488.2
GP9	Down	NA	Missense & synonymous	NM_000174.4
KLK1	Up	NA	Missense & synonymous	NM_002257.3
LMAN1	Up	Cardioembolic, large artery atherosclerosis & small vessel	3’ UTR, synonymous & intron variant	NM_005570.3
MASP1	Up	Cardioembolic, small vessel, large artery atherosclerosis & others	3’ UTR, downstream, upstream, sequence feature, non-coding transcript & intron variant	NM_001879.5 NM_001031849.2 NM_139125.3
PLAUR	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, 5’ UTR & intron variants	NM_002659.3 NM_001005376.2 NM_001301037.1
PRCP	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Downstream, upstream & intron variants	NM_199418.3 NM_005040.3 NM_001319214.1
PROCR	Down	Cardioembolic & others	Missense & intron variant	NM_006404.4
TFPI	Up	NA	Missense variant	NM_001032281.3 NM_001329239.1

Table 7

displays the various genes such as Sec13, Sec24 that are involved in the transport of the AAT protein from production cell to target cell or region. It is important for the intracellular trafficking of the proteins including alpha-1 antitrypsin.
COPII-mediated vesicle transport pathway
Gene	regulation	Stroke subtype (phenotype)	Variants	Protein ID
ANKRD28	Up & down	NA	Missense & synonymous variants	NM_015199.3 NM_00119099.1
AREG	Up	NA	NA	NA
CD59	Up	Large artery atherosclerosis & others	Downstream, intron & 3’ UTR variants	NM_000611.5 NM_001127223.1 NM_001127225.1 NM_001127226.1 NM_001127227.1 NM_203329.2
CNIH1	Up	Others	Intron & 3’ UTR variants	NM_005776.2
CSNK1D	Up	Large artery atherosclerosis	Intron variants	NM_001893.4 NM_139062.2 NR_110578.1
CTSC	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, downstream, 3’ UTR & intron variants	NM_001814.5 NM_001114173.2 NM_148170.4
FOLR1	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, downstream, 5’ UTR & intron variants	NM_000802.3 NM_016729.2 NM_016724.2 NM_016725.2
GOLGA2	Up	Large artery atherosclerosis & others	Intron variants	NM_004486.4
GOSR2	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, missense, 5’ UTR, 3’ UTR, downstream & intron variant	NM_001321133.1 NM_001012511.2 NM_001321134.1 NM_004287.4
SEC13	Down	Large artery atherosclerosis, small vessel & others	Sequence feature, downstream, 5’ UTR & intron variants	NM_001136026.2 NM_001136232.2 NM_030673.3 NM_183352.2
SEC22C	Up	Cardioembolic, large artery atherosclerosis & others	Downstream, 3’ UTR, upstream, synonymous, missense & intron variants	NM_032970.3 NM_001201584.1 NM_004206.3
SEC23IP	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, 5’ UTR, non-coding transcript, synonymous, 3’ UTR, downstream, missense, sequence feature & intron	NM_007190.3
SEC24A	Up	Cardioembolic, large artery atherosclerosis & others	Downstream, upstream & intron variants	NM_021982.2 NM_001252231.1
SEC24C	Down	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, synonymous, downstream, 3’ UTR & intron variants	NM_004922.3 NM_198597.2
SEC16A	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Downstream, 3’ UTR, missense, synonymous, 5’ UTR, upstream & intron	NM_014866.1 NM_001276418.1
STX17	Up	Cardioembolic, small vessel & others	Intron & 3’ UTR variants	NM_017919.2
TRAPPC10	Down	Cardioembolic, small vessel & large artery atherosclerosis	Intron variant	NM_003274.4
TRAPPC2L	Up	Cardioembolic, large artery atherosclerosis & others	Downstream, upstream, 3’ UTR, non-coding transcript & intron variants	NM_001318524.1 NR_134671.1 NM_001318525.1 NM_001318526.1 NM_001318527.1 NM_001318528.1 NM_001318529.1 NM_001318530.1 NM_001318532.1 NM_016209.4

Table 8

represents the genes that participate in the regulation of insulin as well as stroke that impacts various physiological processes, including growth, development, and tissue repair.
Regulation of insulin-like growth factor (IGF) transport and uptake by insulin-like binding factors binding proteins (IGFBPs) pathway.
Gene	regulation	Stroke subtype (phenotype)	Variants	Protein ID
AHSG	Up	Large artery atherosclerosis, small vessel & others	Synonymous & intron variants	NM_001622.2
ALB	Up	Cardioembolic & small vessel	Synonymous & intron	NM_000477.6
CALU	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, intron, 3’ UTR, non-coding transcript, downstream gene variants	NM_001199671.1 NM_001130674.2 NM_001199672.1 NM_001199673.1 NM_001219.4 NR_074086.1
DNAJC3	Up	Large artery atherosclerosis, small vessel & others	3’ UTR & intron variants	NM_006260.4
ENAM	Down	Small vessel & others	Missense, 3’ UTR & intron	NM_031889.2
EVA1A	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Synonymous, 5’ UTR, upstream & intron variants	NM_001135032.1 NM_032181.2
FAM20A	Up	Cardioembolic, large artery atherosclerosis, small vessel & others	Intron, synonymous, 5’ UTR, upstream, downstream & 3’ UTR gene variants	NR_027751.1 NM_001243746.1 NM_017565.3
FN1	Up	NA	Synonymous, missense & splice region variants	NM_212482.2 NM_002026.3 NM_212476.2
FSTL3	Up	NA	Synonymous variant	NM_005860.2
FUCA2	Up	Cardioembolic	Missense & intron variants	NM_032020.4
MATN3	Up	Large artery atherosclerosis & small vessel	Intron variant	NM_002381.4
MGAT4A	Up & down	Cardioembolic, large artery atherosclerosis & others	Downstream & intron variants	NM_012214.2 NM_001160154.1
NUCB1	Down	Large artery atherosclerosis	Intron variant	NM_006184.5
P4HB	Up & down	Cardioembolic, small vessel & others	Synonymous, intron & missense variants	NM_000918.3
PAPPA	Down	Cardioembolic, large artery atherosclerosis, small vessel & others	Intron & 5’ UTR variants	NM_002581.4
PDIA6	Up	NA	Missense & synonymous variants	NM_005742.3 NM_001282704.1 NM_001282706.1 NM_001282705.1 NM_001282707.1
RCN1	Down	NA	Synonymous & missense	NM_002901.2
SERPINA10 Down		Cardioembolic & others	Sequence feature, downstream, 3’ UTR, missense & upstream variants	NM_001100607.2 NM_016186.2
SPARCL1	Up	NA	Missense & synonymous variants	NM_001128310.2 NM_001291976.1
STC2	Up	Large artery atherosclerosis & others	Intron variant	NM_003714.2
TGOLN2	Up	Cardioembolic, large artery atherosclerosis & others	Missense, downstream, 3’ UTR & intron	NM_001206840.1 NM_001206841.1 NM_001206844.1 NM_006464.3

Table 9

depicts the genetic information of gene expression during the transport of the protein AAT from the hepatocytes to the blood stream.
Transport to the Golgi pathway
Gene	regulation		Stroke subtype (phenotype)	Variants	Protein ID
ACTR10	Up		Others	Sequence feature variant	NM_018477.2
ALG2	Up		Large artery atherosclerosis	Upstream & downstream	NM_033087.3
ALG5	Up		Cardioembolic & large artery atherosclerosis	Intron & upstream variant	NM_001142364.1 NM_013338.4
ALG6	Up		Cardioembolic, large artery atherosclerosis & others	Intron variant	NM_013339.3
ALG8	Up		Cardioembolic, large artery atherosclerosis & others	Stop lost, stop retained, upstream, intron & downstream variants	NM_024079.4 NM_001007027.2
AMFR	Up		Cardioembolic & small vessel	Upstream, intron & sequence feature variants	NM_001144.5 NM_001323512.1 NM_001323511.1
ANK2	Up		NA	Missense, synonymous & splice region variants	NM_001148.4 NM_001127493.1 NM_02097.3
ARF3	Up		Small vessel	Intron variant	NM_001659.2
ASGR1	Down		Cardioembolic & others	Downstream, intron & synonymous variants	NM_001671.4 NM_001197216.2
B4GALT1	Up		Cardioembolic & large artery atherosclerosis	3’ UTR & intron variants	NM_001497.3
B4GALT3	Up & down		Cardioembolic & large artery atherosclerosis	Downstream & intron variants	NM_001199874.1 NM_001199873.1
BET1L	Up		Cardioembolic, large artery atherosclerosis & others	Upstream & downstream variants	NM_001098787.1
CALR	Up		NA	Missense, intron & synonymous variants	NM_004343.3 NM_145046.4
COG5	Up		NA	Missense, splice region & synonymous variants	NM_006348.3 NM_181733.2
COPA	Up		Cardioembolic, large artery atherosclerosis & others	Intron, upstream & downstream gene variants	NM_001098398.1 NM_004371.3
COPG1	Up		Others	Missense & intron	NM_016128.3
DCTN2	Up		Cardioembolic & others	Upstream & intron variants	NM_001261412.1 NM_001261413.1 NM_006400.4
DCTN3	Up		Cardioembolic & others	Upstream, downstream & intron variants	NM_007234.4 NM_001281425.1 NM_001281426.1 NM_001281427.1
DCTN5	Down		Others	Intron variants	NM_032486.3 NM_001199011.1 NM_001199743.1
DCTN6	Up		Cardioembolic, small vessel & others	Intron variant	NM_006571.3
DERL2	Up		Cardioembolic, large artery atherosclerosis, small vessel & others	Intron, upstream & downstream gene variants	NM_016041.4 NM_001304777.1 NM_001304779.1 NR_130905.1
DHDDS	Up		Cardioembolic & large artery atherosclerosis	Intron, 3’ UTR, downstream & missense variants	NM_024887.3 NM_001243564.1 NM_001243565.1 NM_205861.2 NM_001319959.1
DYNLL2	Up & down		NA	Intron variant	NM_080677.2
EDEM1	Down		Large artery atherosclerosis, small vessel & others	Synonymous, intron, 3’ UTR variants	NM_014674.2
FUOM	Down		Others	Upstream gene variants	NM_001098483.2 NM_001301827.1 NM_001301828.1
GMPPA	Up		Cardioembolic	Upstream, downstream, 5’ UTR & intron variants	NM_013335.3 NM_205847.2
GOSR1	Up & down		Large artery atherosclerosis & others	Intron variants	NM_004871.2 NM_001007024.1
MAN1C1	Down		Cardioembolic, large artery atherosclerosis, & others	Intron, upstream, synonymous & 3’ UTR variants	NM_020379.3 NM_001289010.1
MGAT3	Down		Cardioembolic, large artery atherosclerosis & small vessel	Downstream, upstream, 3` UTR & intron variants	NM_002409.4 NM_001098270.1
MPDU1	Down		Cardioembolic & others	Downstream, upstream, 3’ UTR & intron variants	NM_001330073.1 NM_004870.3
MPI	Down		Small vessel & others	Intron, synonymous & downstream gene variants	NM_002435.2 NM_001289155.1 NM_001289156.1 NM_001289157.1
NANS	Up		Large artery atherosclerosis	Intron variant	NM_018946.3
NEU1	Up		NA	NA	NA
NEU3	Up		Large artery atherosclerosis	Intron & missense variants	NM_006656.5
OS9	Up		NA	Missense, synonymous & splice region variants	NM_006812.3 NM_001261420.1
PMM2	Up		Cardioembolic, large artery atherosclerosis & small vessel	Sequence feature & intron	NM_000303.2
PSMC1	Up		Cardioembolic, large artery atherosclerosis & others	Upstream & intron variants	NM_002802.2
RENBP	Down		NA	Missense variant	NM_002910.3
RNF103	Up		Large artery atherosclerosis & small vessel	Upstream, downstream, missense, 5’ UTR & intron	NM_005667.3 NM_001198951.1
RNF185	Up		NA	Missense & synonymous variants	NM_152267.3 NM_001135825.1
RPN1	Up		Cardioembolic, large artery atherosclerosis, small vessel & others	Synonymous, 3’ UTR & intron variants	NM_002950.3
RPN2	Up		NA	Missense & synonymous	NM_001324301.1 NM_0011355771.2
RPS27A	Up		Others	Intron variants	NM_001135592.2 NM_002954.5
SPTBN1	Up & down		NA	Missense & synonymous variants	NM_003128.2 NM_178313.2
SRP54	Up		Large artery atherosclerosis, small vessel & others	Upstream, downstream, 5’ UTR, sequence feature, 3’ UTR & intron variants	NM_003136.3 NM_001146282.1
ST6GALNAC2		Up	Others	Intron & 5’ UTR variants	NM_006456.2
ST6GALNAC4	Down		Cardioembolic, large artery atherosclerosis & others	Upstream & intron variants	NM_175039.3 NM_175040.3
ST6GALNAC5 Up			NA	Missense & synonymous	NM_030965.2
ST6GALNAC6	Down		Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, downstream & intron variants	NM_001286999.1 NM_001287001.1 NM_001287003.1 NM_001287000.1 NM_013443.4 NR_104629.1
ST8SIA2	Up		Cardioembolic, large artery atherosclerosis, small vessel & others	Synonymous, splice region, 3’ UTR & intron variants	NM_006011.3
ST8SIA5	Up		Large artery atherosclerosis, cardioembolic & small vessel	Sequence feature, downstream & intron variants	NM_001307986.1 NM_001307987.1 NM_013305.5
STT3A	Up		Cardioembolic, large artery atherosclerosis & others	Synonymous, sequence feature, downstream, 3’ UTR, splice region, intron, upstream gene variants	NM_001278503.1 NM_152713.4 NM_001278504.1
TRIM13	Up		Cardioembolic, large artery atherosclerosis & small vessel	5’ UTR, upstream, 3’ UTR, downstream & intron variants	NM_001007278.2 NM_005798.4 NM_052811.3 NM_213590.2
TUBA1B	Up		NA	Synonymous variant	NM_006082.2
TUBA4A	Up		Cardioembolic, large artery atherosclerosis, small vessel & others	Downstream, upstream & intron variants	NM_006000.2 NM_001278552.1
TUBB1	Down		Others	Missense & synonymous	NM_030773.3
TUBB2B	Down		NA
TUBB4A	Up		Cardioembolic, large artery atherosclerosis, small vessel & others	Upstream, 3’ UTR & intron variants	NM_001289123.1 NM_001289130.1 NM_001289131.1 NM_006087.3 NM_001289127.1 NM_001289129.1
TUBB6	Up		Others	Upstream, downstream & intron variants	NM_001303524.1 NM_001303526.1 NM_001303527.1 NM_001303528.1 NM_001303529.1 NM_001303530.1 NM_032525.2

Table 10

represents the various gene markers that get expressed due to the increased level of cytosolic platelet concentration. This activity has advance events that can lead to stroke.
s
Gene	regulation	Stroke subtype (phenotype)		Variants		Protein ID
A1BG	Up	NA		Missense & synonymous		NM_130786.3
AAMP	Down	Small vessel		Intron variant		NM_001302545.1
ABHD12	Up & down	Cardioembolic, small vessel & large artery atherosclerosis		Downstream & intron variants		NM_015600.4 NM_001042472.2
ANGPT2	Down	NA		Missense, synonymous & splice region variants		NM_001147.2 NM_001118887.1 NM_001118888.1
ANXA5	Up	Cardioembolic, small vessel & others		Intron variant		NM_001154.3
APBB1IP	Up	Cardioembolic, large artery atherosclerosis, small vessel & others		Intron variant		NM_019043.3
ARRB2	Up	Cardioembolic, large artery atherosclerosis & others		Upstream & downstream gene variants		NM_001257329.1 NM_001257330.1 NM_001257331.1 NM_001330064.1 NM_004313.3 NM_199004.1
ATP1B3	Up	Cardioembolic, large artery atherosclerosis & others		Intron variant		NM_001679.3
BRPF3	Up	Others		Sequence feature & intron		NM_015695.2
CBX5	Up & down	Cardioembolic, large artery atherosclerosis & others		Downstream, 3’ UTR, 5’ UTR, sequence feature and intron variants		NM_001127322.1 NM_012117.2 NM_001127321.1
CD36	Up	NA		Missense, synonymous & splice region variants		NM_000072.3 NM_001289911.1 NM_001289909.1 NM_001289908.1
CD48	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Upstream, synonymous & intron variants		NM_001256030.1 NM_001778.3
CD58	Up	Cardioembolic, large artery atherosclerosis & others		Downstream, 3’ UTR & intron variants		NM_001779.2 NM_001144822.1 NR_026665.1
CD63	Up	NA		Missense, synonymous & frameshift variants		NM_001257389.1 NM_001257401.1 NM_001257392.1
CDC42	Up	Large artery atherosclerosis & others		Downstream, upstream & intron variants		NM_001039802.1 NM_044472.2 NM_001791.3
CEACAM3 Up			NA		Missense & synonymous variants	NM_00185.4 NM_001277163.2
CEACAM6 Up			Cardioembolic & others		Sequence feature & intron	NM_002483.6
CFD	Up	Cardioembolic, small vessel & others		Upstream & 3’ UTR variants		NM_001317335.1 NM_001928.3
CFL1	Up & down	NA		Synonymous variant		NM_005507.2
DGKB	Up & down	NA		Synonymous, missense & splice region variants		NM_004080.2
DGKE	Up	Small vessel		3’ UTR variant		NM_003647.2
DOCK1	Up	NA		Synonymous, missense & splice region variants		NM_001290223.1 NM_001380.4
DOCK2	Up	NA		Synonymous, missense & splice region variants		NM_004946.2
DOCK4	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Intron variant		NM_014705.3
DOK2	Down	Cardioembolic & others		Missense, upstream & intron variants		NM_003974.3 NM_001317800.1 NM_201349.2
EHD3	Down	Cardioembolic, large artery atherosclerosis & others		3’ UTR & intron variants		NM_014600.2
ENDOD1	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		3’ UTR & intron variants		NM_015036.2
FCER1G	Up	NA		Missense variant		NM_004106.1
FERMT3	Up	Small vessel & others		Upstream, downstream, synonymous & intron variant		NM_178443.2
FGR	Up	NA		Missense & synonymous		NM_001042729.1
GNA13	Up	Cardioembolic, small vessel & large artery atherosclerosis		Downstream, upstream & intron variants		NM_006572.5 NM_001282425.1
GNAI3	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Upstream, intron, 3’ UTR & 5’ UTR variants		NM_006496.3
GNB5	Down	Cardioembolic, small vessel, large artery atherosclerosis & others		Downstream, upstream & intron variants		NM_016194.3
GNG10	Up	Small vessel & others		Upstream, downstream, 5’ UTR & intron variant		NM_001017998.3
GNG2	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		3’ UTR, upstream & intron variants		NM_001243773.1 NM_001243774.1 NM_053064.4
GNG4	Up	Cardioembolic, large artery atherosclerosis & others		Intron variant		NM_001098721.1 NM_001098722.1
GNG5	Up	Small vessel, large artery atherosclerosis & others		5’ UTR & intron variant		NM_005274.2
GNGT2	Up	Others		Upstream & intron variants		NM_031498.2 NM_001198755.1 NM_001198756.1 NM_001198754.1
GRB2	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Downstream, upstream, 5’ UTR, 3’ UTR & intron variants		NM_002086.4
GTPBP2	Up & down	Small vessel & others		Intron variant		NM_019096.4
GUCY1A2	Up	NA		Missense & synonymous variants		NM_000855.2 NM_001256424.1
GYPB	Up	Large artery atherosclerosis & others		Downstream, upstream & intron variants		NM_002100.5 NM_001304382.1
GYPC	Down	Large artery atherosclerosis & others		Downstream & intron variants		NM_002101.4 NM_001256584.1
HBE1	Up	NA		NA		NA
HRAS	Down	Cardioembolic, small vessel & others		Upstream, downstream & intron variants		NM_001130442.2 NM_001318054.1 NM_005343.3
IRF2	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Intron variant		NM_002199.3
ITGA2	Up	NA		Synonymous, missense & splice region variants		NM_002203.3
ITGA2B	Up	Cardioembolic, small vessel & others		Splice region & intron variant		NM_000419.4
ITGA5	Up	NA		Missense & synonymous		NM_002205.4
ITGAX	Up	Large artery atherosclerosis & others		Upstream & intron variants		NM_001286375.1 NM_000887.4
ITPR1	Down	NA		Missense & synonymous variants		NM_002222.5 NM_001099952.2 NM_001168272.1
ITPR2	Up & down	NA		Synonymous, missense, frameshift & splice region		NM_002223.3
ITPR3	Down	Cardioembolic, small vessel, large artery atherosclerosis & others		Splice region, synonymous, downstream & intron		NM_002224.3
JMJD1C	Up	NA		Synonymous, missense & disruptive in-frame deletion.		NM_032776.2 NM_001282948.1 NM_001318153.1 NM_001322254.1
KIF1C	Up & down	NA		Missense & synonymous variants		NM_006612.5
KIF20A	Up	NA		Missense & synonymous		NM_005733.2
KIF26A	Down	Cardioembolic, large artery atherosclerosis & others		3’ UTR, missense, synonymous & intron variant		NM_015656.1
KIF26B	Down	NA		Missense & synonymous		NM_018012.3
KIF27	Up	Cardioembolic, large artery atherosclerosis & others		Intron variants		NM_017576.2 NM_001271927.1 NM_001271928.1
KIF2C	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Sequence feature, upstream & intron variants		NM_006845.3 NM_001297655.1 NM_001297656.1 NM_001297657.1
KIF3A	Down	Cardioembolic, small vessel & large artery atherosclerosis		3’ UTR, upstream, downstream & intron variant		NM_001300791.1 NM_001300792.1 NM_007054.6
KIF3C	Up	Cardioembolic, large artery atherosclerosis & others		Synonymous & intron variants		NM_007054.6 NM_002254.6
KIF5A	Up & down	Cardioembolic, small vessel & others		Intron variant		NM_004984.2
KIF6	Up	NA		Missense & synonymous variants		NM_145027.4 NM_001289024.1 NM_001289020.1 NM_001289021.1
KIF9	Up	Cardioembolic, small vessel & others		Downstream, sequence feature, upstream, synonymous & intron		NM_001134878.1 NM_022342.4
KLC1	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Intron, downstream & upstream gene variants		NM_001130107.1 NM_005552.4 NM_182923.3
KRAS	Up	Cardioembolic, large artery atherosclerosis & others		3’ UTR & intron variants		NM_033360.3 NM_004985.4
LHFPL2	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		5’ UTR & intron variant		NM_005779.2
MAGED2	Down	NA		NA		NA
MANF	Down	NA		Missense variant		NM_006010.5
MGLL		NA		Missense & synonymous variants		NM_007283.6 NM_001256585.1 NM_001003794.2
MMRN1	Up	Cardioembolic, small vessel & others		Intron variant		NM_007351.2
MPL	Up	Cardioembolic & others		Sequence feature & intron		NM_005373.2
NOS1	Up & down	NA		Missense & synonymous variants		NM_001204218.1 NM_001204213.1 NM_000620.4 NM_001164757.2 NM_014697.3
NOS3	Down	Cardioembolic, small vessel & others		Upstream, missense, synonymous, 3’ UTR, downstream & intron variant		NM_001160109.1 NM_001160110.1 NM_001160111.1 NM_000603.4
NRAS	Down	Large artery atherosclerosis, small vessel & others		Intron variant		NM_002524.4
ORAI2	Up & down	Cardioembolic & others		3’ UTR variant		NM_001126340.2 NM_001271818.1 NM_001271819.1 NM_032831.3
P2RX5	Down	Large artery atherosclerosis		Downstream, upstream & intron variants		NM_001204519.1 NM_001204520.1 NM_002561.3
PCDH7	Down	NA		Missense & synonymous variants		NM_032457.3 NM_032456.2 NM_001173523.1
PDE10A	Up	NA		Missense & synonymous variants		NM_006661.3 NM_001130690.2
PDE11A	Up	NA		Missense, synonymous, frameshift, conservative in-frame insertion variants		NM_016953.3 NM_001077197.1 NM_001077196.1 NM_001077358.1
PDPN	Up	Cardioembolic, large artery atherosclerosis & others		Intron, upstream, downstream, sequence feature & 3’ UTR variants		NM_006474.4 NM_198389.2 NM_001006624.1 NM_001006625.1
PECAM1	Up	Others		Intron & 3’ UTR variant		NM_000442.4
PHF21A	Up	Cardioembolic, large artery atherosclerosis & others		Downstream, 3’ UTR, upstream & intron variants		NM_001101802.1 NM_016621.3
PIK3R1	Up	Cardioembolic, large artery atherosclerosis & others		Upstream, 5’ UTR, 3’ UTR, downstream & intron		NM_181523.2
PIK3R5	Up	Cardioembolic, large artery atherosclerosis & others		Upstream, downstream & intron variants		NM_001142633.2 NM_001251851.1 NM_001251852.1 NM_001251853.1 NM_001251855.1 NM_014308.3
PLA2G4A	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Intron variants		NM_024420.2 NM_001311193.1
PLCG1	Down	Cardioembolic, small vessel, large artery atherosclerosis & others		Upstream, downstream, synonymous & intron variant		NM_002660.2 NM_182811.1
PLEK	Up	Cardioembolic, small vessel & others		Sequence feature, 3’ UTR & intron variants		NM_002664.2
PPP2CB	Up	Small vessel & others		Intron & 5’ UTR variant		NM_001009552.1
PPP2R5B	Down	NA		Missense & synonymous		NM_006244.3
PPP2R5D	Up & down	Cardioembolic, small vessel, large artery atherosclerosis & others		Upstream, downstream & intron variants		NM_001270476.1 NM_180976.2 NM_006245.3
PPP2R5E	Up	NA		Synonymous variant		NM_001282179.1 NM_001282181.1
PRCP	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Downstream, upstream & intron variants		NM_199418.3 NM_005040.3 NM_001319214.1
PRKCB	Up & down	NA		Synonymous & missense variants		NM_002738.6 NM_212535.2
PSAP	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Downstream, 3’ UTR & intron variants		NM_001042465.2 NM_001042466.2
PSG11	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Upstream, downstream & intron variants		NM_002785.2 NM_001113410.1 NM_203287.1
PTGIR	Up & down	Small vessel		3’ UTR & intron variants		NM_000960.3
RBSN	Up	Cardioembolic, large artery atherosclerosis & others		Downstream & intron variants		NM_001302378.1 NM_022340.3 NR_126155.1
RHOA	Up & down	NA		Synonymous variant		NM_001313943.1
RHOB	Up	NA		Synonymous & missense		NM_004040.3
RHOG	Up	Cardioembolic, large atherosclerosis & others		Intron variant		NM_001665.3
S100A10	Up	Cardioembolic & large artery atherosclerosis		Intron variant		NM_002966.2
SCCPDH	Up	Cardioembolic & large artery atherosclerosis		3’ UTR & intron variant		NM_016002.2
SELP	Up	Cardioembolic, large artery atherosclerosis & others		Intron variant		NM_003005.3
SERPINA3 Up			Cardioembolic, large artery atherosclerosis & others		Missense, synonymous & intron variants	NM_001085.4
SERPINB6	Up	Small vessel & others		Downstream, 5’ UTR, upstream & intron variants		NM_001271823.1 NM_001271825.1 NM_001271822.1 NM_001195291.2 NM_001271824.1 NM_001297699.1 NM_001297700.1 NM_001271823.1
SERPINB8	Up	Cardioembolic, small vessel, large artery atherosclerosis & others		Upstream, downstream, 3’ UTR, missense & intron variants		NM_002640.3 NM_001031848.1 NM_001276490.1
SH2B1	Down	Others		Intron variants		NM_001145795.1 NM_001308293.1 NM_001145796.1 NM_001145797.1 NM_001308294.1 NM_001145812.1
SH2B3	Down	Cardioembolic, small vessel, large artery atherosclerosis & others		Intron, upstream, missense, 3’ UTR & downstream variants		NM_005475.2 NM_001291424.1
SRI	Up	Cardioembolic & large artery atherosclerosis		Intron, upstream & downstream gene variants		NM_001256892.1 NM_001256891.1 NM_003130.3 NM_198901.1
STX4	Up	Cardioembolic, small vessel & large artery atherosclerosis		Upstream, 5’ UTR, downstream & intron variants		NM_004604.4 NM_001272096.1 NM_001272095.1
STXBP2	Up	Large artery atherosclerosis, small vessel & others		Sequence feature, downstream, upstream & intron variants		NM_001272034.1 NM_001127396.2 NM_006949.3
TFPI	Up	NA		Missense variant		NM_001032281.3 NM_001329239.1
TLN1	Up	Cardioembolic		Intron & 3’ UTR variant		NM_006289.3
TNFRSF10D	Up & down		Cardioembolic, large artery atherosclerosis & others		3’ UTR & intron	NM_003840.4
TOR4A	Up	Small vessel		Upstream variant		NM_017723.2
TREM1	Up	Others		Downstream, upstream & intron variant		NM_001242589.2 NM_018643.4 NR_136332.1 NM_001242590.2
TRPC3	Up	Large artery atherosclerosis & others		Downstream & intron variants		NM_001130698.1 NM_003305.2
VTI1B	Up	Cardioembolic		Sequence feature & intron		NM_006370.2
WDR1	Up & down	Cardioembolic, small vessel, large artery atherosclerosis & others		Sequence feature, 5’ UTR, upstream, downstream & intron variants		NM_017491.3 NM_005112.4
YWHAZ	Up	Cardioembolic & others		Downstream & intron variants		NM_001135699.1 NM_001135702.1 NM_001135700.1 NM_001135701.1 NM_145690.2

There are various genes such as TUBA1A, COG5, KLC1 as well as SERPINA1 that span more than one pathway, in this case, the gene has been annotated once since its genetic differential expression in relation to the stroke still remains the same.

The stroke has been classified into cardioembolic, large artery atherosclerosis, small vessel occlusion and others which include those with determined etiology and those with undetermined etiology. There have been genetic variants identified in relation to the stroke as explained below;

Downstream and Upstream gene variants

Downstream variants are found after the 3-prime (3’) end of a gene while upstream variants are found before the 5-prime (5’) end. They are found outside the gene and potentially may affect the gene expression.

3’ UTR and 5’ UTR variants

UTR describes the untranslated regions of a gene and they may flank the coding region of a gene but, not into translated protein and this may affect gene regulation.

Sequence feature variants

This term exploits any variation that affects a recognizable sequence element in DNA sequence such as enhancers, promoters, and regulatory motifs.

Intron variants

These are variations that occur in the non-coding regions of a gene when being spliced out of the mature messenger RNA (mRNA) molecule.

Synonymous variant

This is a kind of variant in which the single nucleotide polymorphisms (SNPs) do not affect the amino acid sequence of encoded protein to change.

Missense variants

Here, the SNPs change the amino acid sequence of the protein and the protein function can vary depending on the specific amino acid changed

Frameshift variants

It involves the insertion and/or deletions of nucleotides which disrupts the reading frame of a gene leading to the production of a non-functional protein.

Splice region variants

These variations occur near the splice junctions of the gene between the exons and introns and they can affect fate of a pre-mRNA splicing, potentially leading to formation of an abnormal protein.

Conservative in-frame insertions & Disruptive in-frame deletion variants

The insertions or deletions of nucleotides which do not change the reading frame of the gene but, can still affect the protein function and sequence. Conservative insertions are when nucleotide bases are added in the multiples of three while the adding or deleting of nucleotides not in multiples of three is the disruptive.

4.1 Discussion

Lung fibrosis pathway. The regenerative process following the damage of epithelial cells of the lung tissue especially at the alveolar region can lead to conditions known as pulmonary fibrosis (Yue et. al.,2010). These are usually induced by environmental and smoking-related activities. The process of replacing the damaged cells in order to maintain a barrier function and integrity as well as prevent blood loss involves clotting factors under a clotting cascade such as thrombin. With repeated cycles of damage and repair, there is development of chronic inflammation due to the neutrophilic response as was identified by (Gaggar et. al, 2015) that neutrophil elastase promotes pro-MMP9 and inhibits the TIMP1 hence disrupting the balancing of protease and antiprotease equilibrium. The occurrence of lung fibrosis is due to increased and over-activation of parenchymal cells that act to elucidate wound healing, but eventually form excessive matrix at the alveolar region distorting the lung tissue architecture.

The genes such as Family with sequence 13 member A (FAM13A) gene that participates in cell proliferation due to its isoform 1 and 2. Isoform 2 is down-regulated by the Interleukin-1 Beta (IL-1B) and transforming growth factor (TGF-B1) where it is involved in the regulation of mesenchymal transition. The CEBPB gene expressed in parenchymal and mesenchymal cells has a transcriptional role to regulate between anti-inflammatory and pro-inflammatory signals during tissue repair, through the regulation of extracellular matrix degradation [15]. The CC chemokines such as eosinophil chemotactic factor CCL11 that binds to its cognate receptors CCR3 highly expressed in eosinophils and neutrophils have been associated with progressive pulmonary infiltration [9]. Similarly, CCR2 expression correlates with the increased fibrosis since its attenuation exhibits reduced fibrosis as evidenced [20].

Embryonic and induced pluripotent stem cells and lineage-markers pathway. In this pathway, genes such as Activating Transcription Factor 2 (ATF2) which is a cAMP-dependent factor can translocate to the cytosol due to exposure to genotoxic stress thus impairing with mitochondrial-based cells leading to their death especially due to hypoxic stress [13]. The activity of ATF2 is regulated by the SOX family that includes SOX9, SOX1, SOX5 and others. The oncogenic LIM-Only transcription factor 2 (LMO2) expression in T-cells especially those still immature leads to the initiation of leukemia by providing self-renewal property through which the reactivation of hematopoietic stem cells-specific gene [5], reducing the number of oxygen-carrying cells (red blood cells), white blood cells and/or platelets eventually decreasing the oxygen supply to body tissues and brain, in particular.

On the other hand, LIMO4 has a regulation activity of lung epithelial cells proliferation especially in adults, however, it should be regulated also as it might contribute to cancer development [25]. GATA2 (Zhou et al.,2019) enhances the endothelial-to-hematopoietic transition (EHT) and also generation of hematopoietic stem cells (HSCs), therefore acting as a regulator of normal and leukemia cells. It also mediates the formation of more red blood cells. CXCR4 gene [19], upon expression, provides instructions for making receptor proteins that bind with ligands to stimulate multiple signaling pathways that elucidate hematopoiesis. The receptor disrupts the blood-brain barrier and its down regulation inhibits brain metastasis formation. Hepatocyte nuclear factor 1-beta (HNF-1B) provides instructions for the formation and development of the kidney, pancreas and liver of an embryo (Yu et al., 2015). Any implications relating to liver’s failure to produce normal AAT may have relation to HNF-1B expression.

P73 transcription factor network pathway involves a number of genes that coordinate with p73 to regulate its activity. It is a complex network that involves regulation of cellular activities/processes such as cell cycle arrest, injured/damaged cells. The genes interacting with p73 have, among other functions, signaling duties, cell cycle regulation and therefore when the cell growth is not controlled to normal extent, there might develop cancer. Genes like HSF1 [8 & 24] act to protect brain cells through promoting expression of heat shock proteins that safeguard the cell against stress-induced damage while RELA contributes to the regulation of inflammation in the brain regions [2], which also promote cell survival, unlike genes such as BAK1, BAX and others that participate in cell’s apoptosis.

Nuclear receptors meta-pathway includes proteins that regulate expression of genes, usually activated by specific molecules such as hormones, lipids and xenobiotics binding. These are so important for cell development and differentiation [21]. Any sort of dysfunction may lead to metabolic syndrome, cancer and most importantly to our topic, neurological disorders. The ALAS1 gene responsible for heme synthesis [11], an essential molecule for oxygen transport in red blood cells and therefore low level of heme molecule correlates to low levels of oxygen supply to tissues, and if this happens in the brain region, ischemic stroke can be induced. APOA gene family also has an indirect regulation of the risk of stroke due to their role in metabolism of triglycerides including cholesterol. Mutations in these genes such as APOA5, APOA2 can result to increased cholesterol levels which hinder vascular transport of blood and oxygen [16].

Diseases of hemostasis pathway involves the genes whose expression can regulate hemostasis diseases occurrences that can increase the risk of stroke through affecting the blood clotting process. When the blood vessel gets injured, and there is a rapid formation of blood clots due to inherited or acquired factors, the clots may potentially block blood vessels leading to reduced blood flow towards the brain and consequential low oxygen supply hence stroke occurrence. Additionally, disorders/diseases like sickle cell disease and thrombocytosis may lead to abnormal functioning of platelets that block the cerebral arteries spearheading stroke outbreak [7].

(Coat Protein complex II) COPII-mediated vesicle pathway as well as transport to the golgi and subsequent modification pathway [10] are not directly connected to the stroke, however, the proteins of genes such as Sec24, Sec23 and others are involved in protein trafficking from the production cell to the target cells – in this case alpha-1 antitrypsin is formed in the liver and transported to the bloodstream. Common issues that might happen during transport of the proteins such as protein unfolding that may lead to loss of function due to post translational modifications, impaired targeting of protein that helps it to be easily recognized, blockage of transport vesicles due to mutations in vesicle-forming proteins, all can contribute to the improper functioning of the protein - if not reduced function levels/activity [6]. And the moment the functioning levels of AAT are reduced in the body, the risks of ischemic stroke are increased.

Response to elevated platelet cytosolic Ca²⁺ pathway exhibits the role of increased platelet cytosolic Ca²⁺ in activation of clotting process whereby platelets undergo shape change releasing clotting factors, then clump together to form an aggregate that prevents bleeding [23]. When the abnormal blood clot is formed within the vessels supplying the brain, they tend to block those arteries leading to a decrease in blood flow as well as reduced oxygen supply leading to increased potentiality of ischemic stroke occurrence.

Insulin-like growth factors (IGFs) and their binding proteins [14] are essential in the regulation of the cell survival, growth, and also differentiation of cells providing neuroprotection since the IGFBPs bind to IGFs to influence their transport through the body to the target tissue. The decrease in levels of IGFs and signaling might lead to death of neural cells and brain tissue damage that may result into stroke.

Neutrophil degranulation pathway describes the process in which neutrophils release granules containing various substances. Among the granules, are the azurophil granules that contain the neutrophil elastase enzyme, and their release leads to subsequential discharge of the enzymes into the bloodstream [12]. The enzymes digest the elastin of the lung tissue decreasing the permeability of oxygen leading to less oxygen supply to body cells, and in particular to the brain cells which in the due course may die contributing to the stroke.

The ischemic stroke occurs when blood clot blocks the blood flow to the parts of the brain leading to cell death and tissue damage. The genes involved in inflammation, cell death and tissue repair have a connection to the regulation of the stroke either upwards or downwards. These genes might be either involved in promoting cell survival due to low levels by limiting brain tissue damage or influence genes that are involved in repairing damaged brain tissue and/or promoting growth of new neurons to express themselves. They also may be involved in anti-inflammatory processes that induce stroke-based inflammation.

The variations that happen in the SERPINA1 gene lead to the formation of less normal and more abnormal alpha-1 antitrypsin which leads to deficiency of the protein that eventually results in the increased activity of the neutrophil elastase on the elastin of lung tissues making the permeation of oxygen to bloodstream reduced, and eventually stroke outcome. The studies indicate that people who have relatives with a background of stroke might, though to a lesser extent, contract the stroke and therefore, their continuous health checkup about the stroke traits is highly advisable.

Our future prospective suggests the invention of nano-biosensors that can track the activity of various genes that relate to stroke in real time that the person can easily be identified of any potential development of stroke earlier so that the necessary medical attention can be accorded to prevent the outbreak of the stroke.

In medical settings of future prospectives, these identified gene markers can be employed to diagnosis, track or even treat the stroke patients. Considerations of therapeutical or endogenous regulatory mechanisms such as endogenous attenuators of fibrosis like IL-13Rα2 and LAP (latency-associated protein) that regulate (through deactivation) the fibrosis pathway thereby acting as a negative feedback mechanism, once the desired outcome has been attained. It also regulates the expression of TGF-βs.

Author Contribution

Author Lakshmi. S. led the research, Lusiba .S wrote the manuscript and also other authors contributed equal efforts to the accomplishment of the work. All authors reviewed the manuscript and agreed to the publication of the same.

Data Availability

Data is provided within the manuscript.

Disclosures and Financial support declaration

NONE

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No competing interests reported.

Insilico Analysis of Alpha-1 Antitrypsin Pathway in Ischemic Stroke Patients

Status:

Version 1

Abstract

Figures

1. Introduction

1.1 Sub types of ischemic strokes

1.2 Alpha-1 antitrypsin role in ischemic stroke

1.3 Serpina1 variants

2. Literature review

3. Methodology and tools used

3.1 Materials

3.2 Tools used

3.3 Methods and protocol

4. Results

4.1 Discussion

5. Conclusion

6. Scope of future work

Declarations

Author Contribution

Data Availability

References

Additional Declarations

Status:

Version 1