Development and validation of a novel nomogram to predict the impact of the polymorphism of the ICAM‐1 gene on the prognosis of ischemic cardiomyopathy

The current study investigated the association between polymorphisms of the ICAM‐1 gene and prognosis of Ischemic cardiomyopathy (ICM), and developed a prognostic nomogram for ICM on the basis of ICAM‐1 gene variants. The current study included totally 252 patients with ICM. In addition, PCR‐RFLP (polymerase chain reaction‐restriction fragment length polymorphism) was used to genotype SNPs in the ICAM‐1 gene in the patients. Later, the nomogram model was built by combining clinical data and ICAM‐1 gene variants. This study used the least absolute shrinkage and selection operator (LASSO) regression model to optimize feature selection into an ICM prognostic model. Furthermore, multivariate Cox‐regression was applied to build the prognostic model, which included clinical and gene features chosen by the LASSO regression model. Following that, the receiver operating characteristic (ROC) curve, C‐index, calibration plot analyses and decision curve analysis (DCA) were carried out to evaluate the discrimination ability, consistency, and clinical utility of the prognostic model, and the bootstrap method was adopted for internal validation. predicting factors rs112872667, treating by PCI or CABG, ventricular arrhythmia, left ventricular end‐diastolic diameter (LVDD), use of β‐blockers, systolic blood pressure (SBP), heart rate (HR), and serum sodium were incorporated into the prognostic nomogram. The constructed nomogram performed well in discrimination ability, as observed by the time‐dependent C‐index. Furthermore, as shown by calibration curves, our nomogram's predicted probabilities were highly consistent with measured values. With threshold probabilities, DCA suggested that our nomogram could be useful in the clinic. mutation of rs112872667 have critical predictive value on the prognosis of ICM, ICM patients with the mutant genotype (CT or TT) have higher survival probability than those with the wild genotype (CC). Mutation of rs112872667 in ICAM‐1 gene have critical predictive value on the prognosis of ICM, ICM patients with the mutant genotype (CT or TT) have higher survival probability than those with the wild genotype (CC).

tion into an ICM prognostic model. Furthermore, multivariate Cox-regression was applied to build the prognostic model, which included clinical and gene features chosen by the LASSO regression model. Following that, the receiver operating characteristic (ROC) curve, C-index, calibration plot analyses and decision curve analysis (DCA) were carried out to evaluate the discrimination ability, consistency, and clinical utility of the prognostic model, and the bootstrap method was adopted for internal validation. predicting factors rs112872667, treating by PCI or CABG, ventricular arrhythmia, left ventricular end-diastolic diameter (LVDD), use of β-blockers, systolic blood pressure (SBP), heart rate (HR), and serum sodium were incorporated into the prognostic nomogram. The constructed nomogram performed well in discrimination ability, as observed by the time-dependent C-index. Furthermore, as shown by calibration curves, our nomogram's predicted probabilities were highly consistent with measured values. With threshold probabilities, DCA suggested that our nomogram could be useful in the clinic. mutation of rs112872667 have critical predictive value on the prognosis of ICM, ICM patients with the mutant genotype (CT or TT) have higher survival probability than those with the wild genotype (CC). Mutation of rs112872667 in ICAM-1 gene have critical predictive value on the prognosis of ICM, ICM patients with the mutant genotype (CT or TT) have higher survival probability than those with the wild genotype (CC).

| INTRODUCTION
Cardiovascular disease (CVD) is still a primarily reason for death worldwide. 1 Ischemic cardiomyopathy (ICM), in particular, is a major cause of global prevalence and death. 2 Furthermore, ICM has been detected as the leading reason for CVDs in the United States and the most common risk factor for heart failure (HF). 3 In accordance with the global pandemic, around 26 million ICM cases have cardiac insufficiency, costing global health systems more than $30 billion. 4,5 Furthermore, the mortality rate for cardiac disease cases has been as high as 50% over the last 5 years. 6,7 The initial cause of ICM is the development of atherosclerosis in multi-coronary arteries, particularly the diffusive lesions, and reduced or ceased myocardial blood flow that can generate severe myocardial dysfunction, resulting in heart muscle injury 8,9 and persisting injury.
The content of intercellular adhesion molecule-1(ICAM-1) in blood has previously been proposed as a marker for coronary heart disease (CHD). 2,10,11 ICAM-1, an immunoglobulin superfamily member, is highly denoted in leukocytes and endothelial cells, where it functions as a receptor for the leukocyte integrin lymphocyte functionrelated antigen-1 and Mac-1. 8,12,13 ICAM-1 is an important factor in the pathogenesis of atherosclerosis, exerting critical effects on mononuclear cell recruitment in the vasculature basement membrane. 3,4 Therefore,ICAM-1exerts a vital role in both atherosclerosis and the occurrence of ICM, and in previous study, 14 it is confirmed that the polymorphism of rs5491, located exon2 in ICAM-1 gene, is correlated with ICM.
As previously reported, ICM refers to a disease featured with high morbidity and mortality, and it is costly to the global health system; thus, there is a need to investigate the causes of ICM, as well as predicting factors that have a prognostic value on the prognosis of ICM, and measures to be taken to reduce morbidity and mortality. Although the ICAM-1 gene and its polymorphisms have been linked to ICM, there is no evidence linking it to long-term ICM prognosis. Therefore, we concentrated on determining the relationship between ICAM-1 gene polymorphisms (rs112872667, rs12462944, rs2358581, rs281430, rs281434, rs3093030, rs3093032, rs5030348, rs5030377, rs5491, rs62130660, rs923366) and prognosis of ICM. We also developed a new nomogram model for accurately predicting ICM prognosis based on ICAM-1 gene polymorphisms.

| Ethical statement
Before the start of the current work, all subjects were required to provide informed consent. Our study was designed based on the Helsinki Declaration, and our study protocols were approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University.

| Subjects and study design
From January 2013 to December 2015, participants were recruited from the First Affiliated Hospital of Xinjiang Medical University. The current work enrolled 324 subjects in total, with 252 of them meeting our study eligibility criteria, including 167 alive and 85 dead (cardiogenic death) subjects ( Figure 1). Each participant in the current study had previously received coronary angiography in the hospital or during their most recent hospital stay.
The following criteria were used to make the diagnosis of ICM: (1) coronary angiography revealed >50% luminal stenosis in at least one coronary artery of the leading branch，or have a previous history of coronary artery bypass grafting (CABG) or percutaneous coronary intervention (PCI) (2) N-terminal pro-B-type natriuretic peptide (NT-proBNP) > 125 ng/mL.
The following is the exclusion criteria: Acute decompensated HF; the previous history of unstable hemodynamics; acute myocardial infarction (AMI); liver/kidney/blood/autoimmune diseases; cachexia; noncardiac disorder with a predicted lifespan of <1 year; and those unwilling to participate in this.

| Blood sampling and laboratory tests
On the first day of admission, blood was drawn from each ICM patient and analyzed at the Laboratory of the First Affiliated Hospital of Xinjiang Medical University. White blood cell (WBC), hemoglobin, creatinine (CR), platelet (PLT), high/low-density lipoprotein-cholesterol (HDL-C/LDL-C), blood urea nitrogen (BUN), total cholesterol (TC). In addition, triglyceride levels were all measured (TG).

| Isolation of DNA
Following laboratory tests, this work isolated DNA from venous blood. First, blood samples were centrifuged for 10 min at 1500 rpm with the Eppendorf high-speed centrifuge using the anticoagulant ethylene diamine tetra acetic acid (EDTA) to separate blood cells and plasma. After that, DNA was extracted from peripheral leukocytes with the use of a whole-blood genome extraction kit (Xiamen Kaishuo Biotechnology Corporation, China) and related protocols.
Finally, the extracted DNA sample was stored at 80 C before genotyping.

| Genotyping of the ICAM-1 gene
Of extracted DNA, 1 μL was collected for RNA preparation using specific protocols. Following the detailed instructions, the amplified samples were subjected to SNP genotyping using the SNaPshot multiplex SNP genotyping kit (Application Binary Interface Company, USA).

| Determination of cardiovascular risk factors
Through dividing body weight (kg) by body height squared (m), body mass index (BMI) was calculated. In this study, smokers were defined as those who had smoked for more than 6 months or within the previous 6 months. Drinkers were those who consumed

| Study endpoints in follow-up
The study's endpoint was cardiogenic death during the hospital stay and after discharge, and we recorded the time length from the first diagnosis of ICM to cardiogenic death as the survival time. The patients and their families were contacted by phone. Data, including the dead cases, were obtained through telephone interviews with family members of the deceased patients or through hospital records.
Telephone calls were made three, six, twelve, twenty-four, and sixty months after the initial diagnosis of ICM. Follow-up work was done by trained investigators, and data entry was done by three experienced researchers to ensure data quality. Clinicians trained in systemic data acquisition and event confirmation were in charge of follow-up. if they are not conformed to normal contribution we presented them Median and quartile. Using COX-univariable logistic regression analysis, P < 0.05 was adopted for detecting statistical significance.

| Statistical analysis
Following that, the best predicting factors were chosen using the least absolute shrinkage and selection operator (LASSO) algorithm and adjusted for the decreased high-dimensional data. 17,18 by enrolling significant factors (P < 0.1) from COX-univariable regression.
LASSO regression 19 features with non-zero coefficients were chosen.
Following that, COX-multivariate regression was used to

| RESULTS
The current study included 324 cases in total, with 252 ICM patients included according to the eligibility criteria, of which 167 survived the 60-month follow-up study and 85 died from cardiogenic causes ( Figure 1).
Patients were categorized into two groups on the basis of 60-month follow-up outcomes: survival (n = 167) and cardiogenic death (n = 85). We presented the baseline characteristics of all study subjects, all continues variables were not conform to normal distribution, so presented as median and quartile (Table 1).

| Individualized prognostic model establishment
First and foremost, the prognostic model was created ( The nomogram of the model (Model2), including these variables, was established (Figure 3).

| Nomogramvalidation
We validated this nomogram based on the discrimination ability, As demonstrated in DCA, using our constructed nomogram to predict survival probability yielded a greater net benefit than the "treat none" or "treat all" strategies, demonstrating favorable nomogram clinical utility ( Figure 4F-H). Nomograms are extensively applied as prognostic tools in medicine today. Nomograms rely on user-friendly digital interfaces to achieve enhanced accuracy and to simplify understanding prognosis for better predicting clinical prognosis in CVDs. 23,24 The current study first created a nomogram for predicting ICMc prognosis.

| Follow-up study of the patients
We validated this predictive nomogram in the aspect of discrimination ability, the degree of the consistency between observed and predictive value, clinical utility. Based on AUC value of the ROC and the time-dependent concordance index (C-index), our constructed nomogram demonstrated favorable discrimination capacity, as displayed in Figure 4A, B. Later, the nomogram calibration curves ( Figure 4C-E) were drawn, indicating good consistency between predicted and real values. DCA is a novel test for evaluating a nomogram. 25 According to Figure 4F-H, the DCA demonstrated that using this nomogram to predict the survival probability provides additional benefits over the "treatnone" and "treat-all" strategies, as well as good clinical utility.
We discovered a new predictor factor that can predict the prognosis of ICM and has not been reported in previous studies, which is: variation of rs112872667 in the ICAM-1 gene correlated with ICM F I G U R E 3 Nomogram for evaluation of ICM survival probability. The nomogram was constructed on the basis of genomic and clinical variables, such as polymorphism of rs112872667, treatment by PCI or CABG, complications with ventricular arrhythmias, use of β-blockers, SBP, HR, serum sodium, and LVDD.
prognosis, patients carring the mutant genotype (CT or TT) have higher survival probability than patients with wild genotype (CC).
Although soluble ICAM-1 (sICAM-1) level has previously been linked to ICM and atherosclerosis severity, 26 inhibiting ICAM-1 level can delay atherosclerosis development in apolipoprotein E knockout mice, the relationship of ICAM-1 gene polymorphism with ICM patient prognosis remains unknown. Therefore, our findings are novel and will significantly impact on accurately predicting the prognosis of ICM patients.
Single nucleotide polymorphism is a type of DNA variation that occurs in an individual. 27  According to previous research, 50% of SNPs occur within noncoding regions, 29 rs112872667 SNP is also located in noncoding regions of ICAM-1 gene(intron2), and it shows association with the prognosis of ICM patients, but the mechanism of how rs112872667 play a role on the prognosis of ICM is unclear.
Regardless of How the mechanism is, the mutation rs112872667 is associated with the prognosis of ICM. Based on Cox regression and

AUTHOR CONTRIBUTIONS
Tuersunjiang Naman and Refukaiti Abuduhalike carried out the experiments, analyzed the data, and wrote this paper, which should be considered co-first author.
Aihaidan Abudouwayiti , and Juan Sun did the data collection and follow-up and did extensive literature review.
Ailiman MaheMuti was responsible for the study design, revising the manuscript and streamlining the study. The above authors approved the final version for submission.

ACKNOWLEDGMENTS
Tuersunjiang Naman and Refukaiti Abuduhalike have contributed euqally to this work and share co-first author. The authors thank Tuersunjiang Naman and Refukaiti Abuduhalike for performing the Percutaneous coronary angiography or percutaneous coronary intervention and providing patients with data incorporated in this study.

FUNDING INFORMATION
The current work was funded by grant from the "Key Project of Natural Science Foundation of Xinjiang Uygur Autonomous

CONFLICT OF INTEREST STATEMENT
All authors claimed that there existed no competing interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly available in SNP database at https://www.ncbi.nlm.nih.gov/SNP/snp_viewTable. cgi?handle=LXJMU

ETHICS STATEMENT
The present work gained approval from Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Approval No. 2021D01D17). Individuals who participated into the present work provided informed consents for publishing identifiable data and images contained in the present manuscript.