Identifying single nucleotide polymorphisms (SNPs) and detecting c-reactive protein (CRP) levels associated with posterior circulation ischemia and cerebral infarction

Background: Posterior circulation ischemia (PCI) and cerebral infarction (CI) are major cerebrovascular diseases. Screening potential genetic variations such as single nucleotide polymorphisms (SNPs) in genes involved in the immune system pathway is a reliable assessment of potential risk factors for PCI and CI. Moreover, C-reactive protein (CRP) is recognized as a sensitive biomarker for systemic inammation and secondary lesions related to cerebrovascular diseases, which has a signicant clinical signicance. Methods: We collected whole blood and plasma samples from 39 healthy controls, 23 PCI and 18 CI subjects. Allele-specic polymerase chain reaction (AS-PCR) was used to detect mutations in SNPs. The plasma CRP levels were measured using the newly developed single molecule array (Simoa) method. Results: We detected mutations in three SNPs such as rs3093077, rs3917368 and rs4129267, which are associated with an increased risk of PCI and CI. We also captured a signicant increase in Simoa-measured CRP levels in plasma of patients with PCI and CI, compared to healthy controls. Conclusions: These three identied SNPs can be used as a prediction and evaluation of risk for PCI and CI. Our results demonstrate the high sensitivity of Simoa in detecting plasma CRP levels. Our study highlights that AS-PCR and Simoa technology can be used as sensitive clinical diagnostic methods for PCI and CI. can be used for early screening for disease risk in PCI and CI, in particular rs3917368 with a high risk index. In addition, CRP is one of the potential biomarkers for clinical diagnosis of PCI and CI. Our results indicate the high sensitivity of the Simoa technology in detecting plasma CRP levels. Monitoring the level of CRP is helpful to prevent and control the progression of PCI and CI.


Introduction
Posterior circulation ischemic (PCI) stroke is a clinical syndrome associated with ischemia related to stenosis, in situ thrombosis, or embolic occlusion of the posterior circulation arteries-the vertebral arteries in the neck, the intracranial vertebral, basilar, and posterior cerebral arteries, and their branches (Chen et al. 2018;Murai et al. 2008). Patients with symptomatic vertebral basilar artery stenosis have a 3fold increased risk of stroke after transient ischemic attack (TIA) or mild stroke than patients without stenosis. In addition, cerebral infarction (CI) is a common cerebrovascular disease that can lead to neurological damages such as exercise weakness, speech dysfunction, vision loss, dizziness, severe headache, and even death. Beside of imaging approaches, we lack of characteristic biomarkers for precise diagnostics for cerebrovascular diseases.
C-reactive protein (CRP) is released mainly by the liver in response to interleukin-6 (IL-6) and IL-1 after in ammatory insults (Pepys et al. 2003). As a biomarker of chronic in ammation, the association of CRP expression level with various diseases has been studied. For example, single nucleotide polymorphism (SNP) studies have shown that increased serum CRP levels are associated with CRP rs3093077 (p<0.001) in patients with early (p=0.016) and late (p<0.001) diabetes (Qi et al. 2009). Whole Genome-Wide Association Studies (GWAS) also have identi ed a variant rs11265259, which is associated with CRP levels in 200,000 individuals of European descent (Ra eld et al. 2020). In addition to the SNPs of the CRP gene, some other SNPs of in ammation related genes, such as rs4129267 and rs6734238 of IL-6R and IL-1F10 loci, respectively, also have been found signi cantly associated with coronary heart disease (Sabater et al. 2013). SNP rs3917368 of IL-1B has been shown to be related to thyroid ophthalmopathy in Taiwanese, and related to hay fever in Japanese (Fuji et al. 2018). These studies indicate an essential association of SNPs with in ammatory immune system diseases. However, whether these SNPs are related to CRP levels and are associated with PCI and CI have not been explored.
Furthermore, because CRP is a sensitive biomarker for systemic in ammation for infection, trauma, surgery, burns, tissue reaction infarction, advanced cancer and chronic in ammation, it has a great potential for predicting secondary lesions associated with cerebrovascular diseas (Gabay et al. 1999). However, it has been a challenge to detect accurate levels of CRP in blood samples.
In this study, to establish disease risk of important SNPs of in ammation related genes in PCI and CI, we examined ve SNPs by using allele-speci c polymerase chain reaction (AS-PCR) (Darawi et al. 2013). AS-PCR, also known as ampli cation of refractory mutation system (ARMS), is a method based on polymerase chain reaction and can be used to detect known SNPs (Kwok et al. 2003). AS-PCR has been widely used in many research elds such as hereditary diseases and pharmacogenetics (Chen et al. 2007;Mirasena et al. 2008).Among the ve SNPs, rs3093077 (CRP), rs4129267 (IL-6R) and rs3917368 (IL-1B) displayed a correlation with PCI and CI. Moreover, to detect the concentration of CRP in plasma, we applied the single molecule array (Simoa) technique. Compared with healthy subjects, we detected a signi cantly higher plasma CRP levels in subjects with PCI and CI using Simoa. Moreover, we examined the correlation between SNP mutations and CRP levels. Our results indicate that SNP mutations have a potential in uence on CRP levels and are associated with PCI and CI. Our study also indicates a useful tool of Simoa to detect CRP levels under the disease condition.

Apparatus
The ABI Quant Studio 6 (ABI/USA) has 5-colorxcitation light lters and 5-color detection light lters, which can detect up to 5 colors of uorescence simultaneously, supporting multi-target detection. Consumables such as rapid 96-well (0.1 mL) reaction plate and optical cover lm, 0.1 mL eight-connecting tube, 0.1 mL single tube, and 384-well reaction plate and optical cover lm were used.
In addition, the Simoa HD-1 analyzer (Quanterix©, USA) was used for immunoassay. Immune complexes attached to magnetic beads were captured and individually enclosed in microwells on a Simoa disc. The Simoa disc was manufactured by Sony DADC. Each disc contained 24 array chips and could detect 24 samples. Each chip had 216,000 micropores, and each micropore could hold one magnetic bead with or without an immune complex.

Plasma sample collection
This study was approved by the Institution Ethic Issue Committee of the First Hospital of Quanzhou, Quanzhou, Fujian China (2019-107). Blood samples were collected from healthy, posterior circulation ischemia and cerebral infarction individuals. Plasma was separated through a centrifuge and stored at -80°C until use.
Allele-speci c polymerase chain reaction (AS-PCR) assay DNA was extracted from 200µl venous blood per subject using a DNA extraction kit (Blood Genomic DNA mini kit, CW0541S) according to the manufacturer's protocol. Primer Premier 5 software was used and speci c primers were designed according to the principles of base complementary pairing and mismatch. Primer veri cation was assisted by Sanger sequencing, and genotype was interpreted according to △Ct calculation and analysis. PCR ampli cation was performed in 20µL reactions of 10 pmol/µL of respective primers, 100µM dNTPs, 1 mM MgCl 2 , 1 X PCR buffer, 0.5U Taq Polymerase and 50 ng/µL of DNA samples. DNA was denatured at 95℃ for 30s, followed by 35 cycles of denaturation at 95℃ for 5s, annealing and extension at 60℃ for 30s.

Single nucleotide polymorphisms (SNP) analysis and primer design
We identi ed mutations in both PCI and CI samples by detecting ve candidate SNPs using AS-PCR in combination with uorescence. SNPs such as rs11265259, rs3093077, rs4129267 and rs6734238 are located in the promoter regions of CRP, IL-1F10 and IL-6R genes, respectively, and rs3917368 is located in the coding region of IL-1B gene. According to the sequences of wild-type (W) and mutation (M) of SNPs, speci c ampli cation primers were designed based on the principle of AS-PCR primer design to identify genotypes. Simoa assay Plasma samples were analyzed using the Simoa HD-1 Analyzer according to the manufacturer's instruction. The sample tube was rst positioned and inserted into a channel in a sample holder. The integrated barcode reader read the sample ID barcode as it was inserted into the sample holder. Each sample required two disposable tips, a reaction cuvette and disposable Simoa discs of 24 arrays. When the processing of each sample was complete, the results were computed from the array image le by a software algorithm that includes quality control (QC) checks on quality of the data. The measurement results were automatically calculated and displayed in real time after each sample was processed.

Calculations of odds ratio (OR) values and Pearson correlation coe cient
The potential correlation between genotype and disease was analyzed by OR value calculation. OR and 95% con dence interval (CI) of each genetic model were calculated by random effect model analysis.
Pearson correlation coe cient, also known as Pearson product-moment correlation coe cient, is a linear correlation coe cient. The correlation coe cient is generally expressed as R, which describes the degree of linear correlation between two variables. If R>0, the two variables are positively correlated. If r<0, the two variables are negatively correlated. A larger absolute value of R indicates a stronger correlation.

Statistical analysis
Statistical analysis was performed using Statistical Product and Service Solutions (SPSS) (IBM, Armonk, NY). Unpaired Student's T-test was used for assessing the signi cance of differences between two groups. Graphic processing was performed using GraphPad Prism (GraphPad; La Jolla, CA). All data was presented as mean ± SD, P <0.05 was regarded as being signi cant.

Blood sample collection
Posterior circulation ischemic stroke (PCI) and cerebral infarction (CI) are common cerebrovascularrelated disorders. To determine biomarkers for PCI and CI, we collected whole blood and plasma samples from healthy control and patient subjects. Blood samples were collected from 23 PCI patients, including 11 males and 12 females, aged between 47 and 88 years old, with an average age of 66. In addition, blood samples were collected from 18 CI patients, 13 males and 5 females, aged between 15 and 82 years old, with an average age of 59. Blood samples from 39 healthy controls, including 24 females and 15 males, aged between 16 and 72 years old, with an average age of 43, were served as controls (Table  1). Collected whole blood and plasma samples were stored at -80℃ for further analyses.

Difference of SNP mutations among healthy controls, PCI and CI subjects
To explore the potential association of SNP mutations with the risk for PCI and CI, genotypes of the 5 SNPs were analyzed by AS-PCR in whole blood samples of all subjects (Table 2). We rst analyzed the genomic locations of these SNPs (Figure 1). We found that rs11265259 and rs3093077, rs6734238, and rs4129267 are located in the promoter regions of CRP, IL-1F10 and IL-6R genes, respectively, and rs3917368 is located in the coding region of IL-1B gene (Figure 1). According to the wild type and mutation sequences of these SNPs, we designed speci c ampli cation primers and performed AS-PCR.
We did not detect any mutations for CRP rs11265259 and IL-1F10 rs6734238 in all control and patient samples in this study (Table 2). Interestingly, we detected mutations in the other 3 SNPs among both healthy control and patient samples. We calculated the odds ratio (OR) values for each genotype using SPSS software, and compared the risk estimate data to evaluate the risk genotypes for PCI and CI ( Figure  2, We found that the OR values of CRP rs3093077 were less than 1, suggesting that the C allele of CRP rs3093077 may be a protective factor in reducing PCI and CI risk. However, the OR values of IL-1B rs3917368 and IL-6R rs4129267 in PCI and CI samples were greater than 1, compared to those in healthy controls, suggesting that these SNP mutations may increase the risk of PCI and CI ( Figure 2). Notably, when the homozygous genotype for the TT mutation was present in IL-1B rs3917368, the OR values for PCI and CI subjects were as high as 18.75 and 9, respectively, compared to those in healthy controls, suggesting a great increase of disease risk (Figure 2, Table 3). Our results suggest that these three SNPs can be used to predict and evaluate the risk of PCI and CI.

Signi cant differences in CRP levels detected by Simoa
Because Simoa is a fully automated analysis with higher sensitivity and accuracy compared to conventional methods, we next measured CRP levels in healthy controls, PCI and CI subjects. For the healthy controls, the minimal CRP value was 1.3333 pg/ml, the maximal value was 364.2411 pg/ml, and the average was 44.9273 pg/ml, as measured by Simoa (Table 4). On the other hand, the lowest CRP concentration was measured as 2.5520 pg/ml, the highest was 569.6146 pg/ml, and the average was 159.6868 pg/ml in plasma samples in the CI subjects (Table 4). Moreover, the lowest CRP concentration was 11.3224 pg/ml, the highest was 781.1730 pg/ml, and the average was 281.4760 pg/ml, as measured in plasma samples from the PCI subjects (Table 4). Our Simoa analyses indicate a signi cant increase of CRP concentrations in plasma samples from PCI and CI patients, compared to those from healthy controls.
Moreover, we performed Pearson correlation coe cient analyses based on SNP mutation percentages in each CRP concentration range for each gene. We found that the correlation coe cient R for the IL-1B rs3917368 mutation and CRP levels is 0.81, suggesting a very high correlation between this SNP mutation and CRP levels ( Table 5). The correlation coe cient R for the IL-6R rs4129267 mutation and CRP levels was 0.55, suggesting a moderate correlation between this SNP mutation and CRP levels. The correlation coe cient R for the CRP rs3093077 mutation and CRP levels was -0.28, indicating a negative correlation between them (Table 5).
These results suggest that SNP mutations in IL-1B rs3917368 and IL-6R rs4129267 are positively correlated with CRP levels.

Discussion
Posterior circulation ischemia (PCI) and cerebral infarction (CI) are common cerebrovascular diseases. Accumulating studies have shown that CRP is directly involved in cardiovascular diseases such as in ammation and atherosclerosis. In this study, we have examined SNP mutations that are associated with PCI and CI, detected increased CRP levels in plasmas from PCI and CI patients using a high sensitive method Simoa. Our study con rms CRP as a potential biomarker for cardiovascular disease, further highlights AS-PCR and Simoa as powerful tools in clinical applications.
Precision medicine, applying accurate screening, auxiliary diagnosis and treatment, is becoming a promise direction in medical research and practice. The IL-1 cytokine family consists of 11 members, which are mainly responsible for controlling pro-in ammatory response to tissue damages, and plays a vital role in many autoimmune diseases (Gabay et al. 2010;Palomo et al. 2015). IL-6 is a multi-functional cytokine, and plays an important role in immune regulation, hematopoiesis, in ammation and tumorigenesis (Jones et al. 2018). IL-6 stimulation results in an activation of the JAK/STAT pathway ( Figure 5). Both STAT1 and STAT3 are phosphorylated and are capable of forming homologous and heterologous dimers, which lead to their localization and subsequent regulation of transcription of their target genes, and in turn affect CRP levels ( Figure 5). Thus, IL-6, IL-1, and CRP are recognized as ideal targets for diagnosis of disorders related to in ammation. Detecting mutations in SNPs and exploring their associations with disease risk are crucial in early diagnostics of various diseases. In this study, we have assessed SNP mutations and identi ed potential risks in genes involved in in ammation, which are associated with PCI and CI.
Identifying and applying biomarkers associated with diseases are critical for early disease diagnosis and prognosis (Rifai et al. 2006). Proteins and nucleic acids are currently the most broadly used biomarkers in basic research and clinical application. The AS-PCR can conveniently and quickly detect mutations at speci c sites of genes. The analysis and interpretation of genotypes by △Ct are accurate and reliable, and the overall cost is far lower than that of high-throughput sequencing (Lang et al. 2011). Therefore, AS-PCR can be used as a powerful genotyping identi cation tool widely used in risk assessment of speci c diseases.
Moreover, because the protein content is much higher than the nucleic acid content, it is becoming a reliable clinical diagnosis target (Simon 2011). Enzyme linked immunosorbent assay (ELISA) uses immune reaction to provide qualitative and quantitative measurement of a target protein, and has been a well-accepted method and a powerful tool in research labs and clinic (Engvall et al. 1971). However, the requirement of a relatively higher protein level has hindered its usage to detect subtle differences among samples and a low level of proteins. The recently developed Simoa technology has overcome the drawbacks of ELISA. Simoa combines automated ELISA with single molecule array termination to enable single molecule sensitivity and multiplex analysis (Chang et al. 2012;Yeung et al. 2016). Simoa has been used to quantify levels of biomarkers associated with diseases such as neurological disorders (Hansson et al. 2017). For example, levels of amyloid-β (Aβ42) peptide, tau, phosphorylated neuro lament (pNfH), and neuro lament light chain (NfL) have been measured using the Simoa technology (Bjerke et al. 2016;Zetterberg et al. 2011;Mattsson et al. 2016;Rojas et al. 2018;Kasai et al. 2017;Wilke et al. 2019;Kuhle et al. 2019).
As a newer platform, the Simoa technology has demonstrated a great potential to sensitively quantify proteins with a hint amount. However, the current expensive reagents and equipment have limited the broad use of the Simoa technology in research, let along in clinical application. Nevertheless, the fast improvement of the Simoa technology should expand its ground. Along with the ELISA method, Simoa will become a useful tool for clinical diagnosis.

Conclusion
In summary, SNP screening plays an important role in early detection of the risk assessment of PCI and CI. Three SNPs, including rs3093077, rs3917368 and rs4129267, can be used for early screening for disease risk in PCI and CI, in particular rs3917368 with a high risk index. In addition, CRP is one of the potential biomarkers for clinical diagnosis of PCI and CI. Our results indicate the high sensitivity of the Simoa technology in detecting plasma CRP levels. Monitoring the level of CRP is helpful to prevent and control the progression of PCI and CI. Consent for publication -Not applicable.

Availability of supporting data -
The data sets in this study are available from the corresponding author upon request.
Competing interests -Wilke C, Pujol-Calderón F, Barro C, Stransky E, Blennow K, Michalak Z, et al. 2019. Correlations between serum and CSF pNfH levels in ALS, FTD and controls: a comparison of three analytical approaches.  The OR values of different genotypes were calculated in samples of the healthy controls (HC), posterior circulation ischemia (PCI) and cerebral infarction (CI) subjects. cerebral infarction (CI) subjects, including males and females of different ages. The Simoa was measured as pg/ml. Unpaired Student's t-test was used for assessing the signi cance of differences between two groups. ***: P < 0.001.