1 Original Article

2
3 Correlations between the TIM-4 gene polymorphism and coronary atherosclerotic
4 heart disease
5
6 Dejian Cai1, Wen Liu2, Yuemin Feng3, Jie Xiang4, Liang Tian4, Hao Wei5, Shuai
7 Zhang6, Qiang Zhu3, Xiaohong Liang2, Chunhong Ma2, Lifen Gao2
8
1
9 Department of Clinical Laboratory, Shandong Second Provincial General Hospital,
10 Shandong University, Jinan, China; 2Department of Immunology, Key Laboratory for
11 Experimental Teratology of Ministry of Education, Shandong Provincial Key
12 Laboratory of Infection & Immunology, School of Basic Medical Sciences, Shandong
13 University, Jinan, China; 3Shandong Provincial Hospital Affiliated to Shandong First
14 Medical University, Jinan, China; 4Department of Clinical Laboratory, Shandong
15 Second Provincial General Hospital, Jinan, China; 5Out-Patient Department, Shandong
16 Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China;
6
17 Department of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical
18 University, Xuzhou, China
19
20 Cai et al. Correlations between the TIM-4 gene polymorphism and CHD
21
22 Contributions: (I) Conception and design: Lifen Gao,Dejian Cai; (II) Administrative
23 support: Wen Liu, Yuemin Feng; (III) Provision of study materials or patients: Hao
24 Wei, Shuai Zhang; (IV) Collection and assembly of data: Qiang Zhu, Xiaohong Liang,
25 Chunhong Ma; (V) Data analysis and interpretation: Dejian Cai,Jie Xiang, Liang Tian;
26 (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.
27
28 Correspondence to: Dr. Lifen Gao. Department of Immunology, Key Laboratory for
29 Experimental Teratology of Ministry of Education, Shandong Provincial Key
30 Laboratory of Infection & Immunology, School of Basic Medical Sciences, Shandong
31 University, 44 Wenhua Xi Road, Jinan 250012, China. Email: glfflg@sdu.edu.cn.
32 Abstract
33 Background: Coronary atherosclerotic heart disease (CHD) is a chronic inflammatory
34 disease, and the monocyte-macrophage system plays a complex and decisive role in the
35 development of atherosclerotic plaques.T-cell immunoglobulin and mucin domain
36 containing 4 (TIM-4), acting as a receptor for phosphatidylserine (PS), promotes the
37 phagocytosis of apoptotic cells. However, the relationship between the TIM-4 gene
38 polymorphisms and CHD remains unclear at present.
39 Methods: Peripheral blood samples and sera of patients with CHD and healthy controls
40 were collected, and an analysis of age, sex, smoking status, drinking status, serum lipid
41 level, and other related data was performed. Specific primers were designed according
42 to rs7700944 and rs6882076 polymorphisms, and genotyping was performed using
43 real-time quantitative polymerase chain reaction (PCR) method to calculate genotype
44 and allele frequency. The level of soluble TIM-4 (sTIM-4) in the serum of the healthy
45 control group and patients with CHD was detected by enzyme-linked immunosorbent
46 assay (ELISA). The difference in genotype and allele frequencies of rs7700944 and
47 rs6882076 was compared between the healthy control group and the CHD group. The
48 differences of serum sTIM-4 levels in the CHD and healthy controls were analyzed, as
49 well as those in CHD patients with different genotypes at rs7700944. The relationship
50 between rs7700944 genotypes and hypertension, diabetes, blood lipids, and coronary
51 scores in CHD were further evaluated.
52 Results: The levels of sTIM-4 in serum of patients CHD were significantly lower than
53 those of healthy controls. The TIM-4 gene rs6882076 was not associated with CHD,
54 while rs7700944 was significantly correlated with CHD, with the AA genotype being
55 less sensitive to CHD. The level of sTIM-4 in homologous serum of rs7700944 in
56 patients with CHD was significantly higher than that in patients with the GA genotype,
57 further indicating that the homozygous TIM-4 gene rs7700944 AA may be a protective
58 gene for CHD. As for the coronary score, the AA genotype had a higher score than did
59 the GG type.
60 Conclusions: sTIM-4 levels in patients with CHD were lower than those in healthy
61 controls, which indicates that sTIM-4 might prevent the development of CHD.
62 Homozygous TIM-4 gene rs7700944 AA may exert a protective effect against CHD.
63 Keywords: T-cell immunoglobulin and mucin domain containing 4 (TIM-4); coronary
64 atherosclerotic heart disease (CHD); single-nucleotide polymorphism (SNP); soluble
65 TIM-4 (sTIM-4)
66 #Introduction
67
68 Coronary atherosclerotic heart disease (CHD) is a complex disease that is caused by
69 the long-term interaction between multiple genes and environmental factors and which
70 seriously threatens human health (1). Dyslipidemia (such as hypercholesterolemia and
71 hyperlipidemia), hypertension, diabetes, impaired glucose tolerance, and smoking,
72 among other factors, contribute to CHD pathogenesis (2). Generally, CHD is a chronic
73 inflammatory disease, and the presence of lymphocytes in the lesions suggests that
74 inflammation and immune responses play an important role in the development of
75 atherosclerosis (3). It has been found that cytokines and various immunoregulatory
76 molecules are also involved in the development and progression of CHD (4,5). Recently,
77 a series of CHD-associated susceptibility genes have been discovered (6,7), and
78 genome-wide association studies (GWAS) have revealed that more than 60 single-
79 nucleotide polymorphisms (SNPs) are associated with CHD (8).
80
81 The T cell immunoglobulin and mucin domain (TIM) family includes 3 members: TIM-
82 1, TIM-3, and TIM-4. TIM-1 and TIM-3 are mainly expressed in T-cells, while TIM-4
83 is mainly expressed in antigen-presenting cells (APCs), especially in macrophages and
84 dendritic cells (9,10). TIM-4 can promote T-cell proliferation and activation by binding
85 to its ligand TIM-1 (11). Recent studies have reported that TIM-4 blockade aggravates
86 the progression of atherosclerosis, which might be related to the inhibition of apoptotic
87 cell phagocytosis and T-cell activation (7). We have found that overexpression of TIM-
88 4 inhibits the expression of major histocompatibility complex (MHC) class II molecules,
89 costimulatory molecules CD80 and CD86, and the production of inflammatory factors,
90 such as tumor necrosis factor alpha (TNF-α) of macrophages (12). In addition, we have
91 reported that TIM-4 can inhibit the production of nitric oxide (NO) in macrophages via
92 the nuclear factor kappa beta (NF-κB) signaling pathway (13,14). In this study, we
93 further examined the relationship between TIM-4 and CHD.
94
95 #Methods
96
97 ##Study population
98 There were 499 patients CHD and 500 healthy people included in this study, and a case-
99 control design was used to investigate the correlation between soluble TIM-4 (sTIM-4)
100 and CHD. The protocol for the study was approved by the ethics committee at the
101 School of Basic Medical Sciences, Shandong University (No. ECSBMSSDU2019-1-
102 010).
103
104 ##Enzyme-linked immunosorbent assay (ELISA)
105 The concentration of sTIM-4 in the serum was measured using ELISA kits (Cloud-
106 Clone Corp., USA).
107
108 ##DNA extraction
109 The whole genomic DNA in peripheral blood leukocytes was extracted using a Whole
110 Blood Genomic DNA Extraction Kit (catalog no. DP318; Tiangen Biotech, USA).
111
112 ##SNP detection and typing
113 For quantitative polymerase chain reaction (qPCR) amplification and sequencing, a pair
114 of sequencing primers were designed based on the SNP rs6882076 and rs7700944
115 nucleic acid sequences published in the TIM-4 gene sequence in the National Center
116 for Biotechnology Information (NCBI) database. Three SNP-typing primers (2 specific
117 typing and 1 common corresponding primer) were designed for qPCR amplification
118 and typing. To verify the specificity of the primers, the designed primers were aligned
119 with human genome-wide sequences using NCBI’s Basic Local Alignment Search Tool
120 (BLAST) program, and the verified primers were synthesized by Jinweizhi
121 Biotechnology (Suzhou, China).
122
123 The rs7700944 sequencing primers were used in the following order:
124 F: 5'-CTCGGCCCTTGTGTGCTTAT-3';
125 R: 5'- ATGCTTGGGTGGGTGTCTTT-3'.
126
127 The rs7700944 SNP typing primers were used in the following order;
128 Wild type: F: 5'-GAGGCAAGACAATAAAGTGTGTAAG-3';
129 Mutant type: F: 5'-GAGGCAAGACAATAAAGTGTGTAAA-3';
130 Common downstream: R: 5'-TGGCTCAACGTGTCTAGTGC-3'.
131
132 The rs6882076s sequencing primers were used in the following order;
133 F: 5'-AAACCCCCAGACCAGTCTCT-3';
134 R: 5'-GTGGCACATGCACAGGTTAT-3'.
135
136 The rs6882076SNP typing primers were used in the following order;
137 Wild type: F: 5'-AATGCTGACAGGGTGGGACTCC-3';
138 Mutant type: F: 5'-AATGCTGACAGGGTGGGACTCT-3';
139 Common downstream: R: 5'-ACAAAGAAGCACAAGTATGTCCACG-3'.
140
141 A PCR kit (Talent, China) and a Bio-Rad PCR instrument were used for the sequencing
142 primers amplification under the following conditions: predenaturation at 95 ℃ for 10
143 min; denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 30 s, and extension at 72 ℃
144 for 30 s and for 30 cycles; and extension at 72 ℃ for 10 min. Following this, 1.5%
145 agarose gel (Solarbio, China) containing Gel Red (1:10,000) was prepared, and the
146 result was viewed by an automatic gel imager.
147
148 ##qPCR SNP analysis
149 The total DNA samples were extracted, and the typing primers were added according
150 to a 25-μL total reaction system as follows: 12.5 μL of 2× Talent qPCR PreMix; 0.5 μL
151 of forward primer ; 0.5 μL of reverse primer ;1 μL of DNA template; 10.5 μL of double-
152 distilled water; and amplification instrument, StepOnePlus Real-Time
153 PCR(Talent,China) . qPCR was performed under the following conditions: 5 min at
154 95 ℃, followed by 40 cycles of 15 seconds at 95 ℃, 20 seconds at 60 ℃, and finally
155 30 seconds at 72 ℃. The melt curve was then selected.
156
157 ##Statistical analysis
158 Statistical analysis was performed using SPSS 19.0 software (IBM Corp., USA). The
159 data were processed and analyzed using the Graph-Pad Prism 6.0 (GraphPad Software,
160 USA). Measurement data that obeyed or approached a normal distribution are
161 expressed as mean ± standard deviation (SD). The comparison between the two groups
162 was performed using an independent samples t-test, and the comparison between the
163 multiple sets of data was performed by with one-way analysis of variance (ANOVA).
164 The correlation between sTIM-4 and serum lipids and Gensini score was analyzed with
165 Pearson correlation coefficient, with a P value <0.05 being considered statistically
166 significant.
167 #Results
168
169 ##The levels of sTIM-4 in serum of patients with CHD were significantly lower than
170 those of healthy controls
171 In order to explore the relationship between serum sTIM-4 levels and CHD, we
172 compared the general conditions of the healthy control group and the CHD group. The
173 results showed that patients with CHD were relatively older compared with healthy
174 controls and that the proportion of smoking and drinking was also relatively higher. In
175 terms of lipid metabolism, patients with CHD had higher triglyceride (TG) and high-
176 density lipoprotein cholesterol (HDL-C) compared with healthy controls, and there
177 were no statistical difference between low-density lipoprotein cholesterol (LDL-C) and
178 total cholesterol (TC) (Table 1). Next, we further tested serum sTIM-4 levels and found
179 that serum sTIM-4 levels were significantly lower in patients with CHD than in healthy
180 controls (Figure 1, P<0.001), suggesting that sTIM-4 may play a role in CHD.
181
182 ##Allele-specific quantitative real-time PCR (AS-qRT-PCR) genotyping results and
183 PCR amplification and sequencing
184 The SNP detection method we designed is an AS-qRT-PCR system. Two specific typing
185 primers and a common primer were designed by using the AS-qRT-PCR principle. The
186 last base of the specific typing primer 3' untranslated region (3' UTR) was matched to
187 the wild type and mutation type sites of the SNP, respectively. AS-qRT-PCR-specific
188 primers do not need the introduction of a mismatch, which reduces the difficulty of
189 primer design and increases the success rate of PCR amplification. The classified
190 primers were matched with common primers to form the fluorescence quantitative
191 detection system of wild type and mutant type, and a fluorescence quantitative PCR
192 analyzer (Bio-Rad, USA) was used for amplification. The melting temperature and
193 graphics of the melting curve were used to distinguish specific amplification from
194 nonspecific amplification and invalid amplification. Specific amplification has a single-
195 peak melting figure and a fixed melting temperature, nonspecific amplification can
196 have a similar melting temperature or different melting temperature, invalid
197 amplification has no consistent melting temperature and different melting curve.
198 Meanwhile, sequencing primers of the same SNP locus were designed, and part of the
199 samples were taken to verify the accuracy of AS-qRT-PCR genotyping results with the
200 sequencing method.
201 Taking rs6882076 as an example, the amount of complement DNA (cDNA) template is
202 the same for both the wild-type and mutant-type tubes. After qRT-PCR amplification,
203 if the 2 Δcycle threshold (ΔCT) values are the same or similar (the difference between
204 the 2 is less than 2, the majority is less than 1) and there is a single peak melting pattern
205 and a specific melting temperature, the specimen genotype is heterozygous (Figure 2).
206 If the ΔCT value of the wild-type amplification tube is smaller than that of the mutant
207 amplification tube (the experimental difference is greater than 6 ),, the genotype of the
208 specimen is wild homozygous.Otherwise, the opposite genotype is homozygous for
209 mutation (Figure 3). Fifteen samples were collected at rs7700944 and rs6882076,
210 respectively. PCR amplification, electrophoresis, and sequencing were performed by
211 sequencing primers. The results of genotyping and AS-qRT-PCR genotyping were
212 consistent with the sequencing of the corresponding specimens. Figures 4,5 show the
213 results of the electrophoresis and genotyping of rs7700944 and rs6882076 sequencing
214 and typing. The PCR amplification band at the expected molecular weight position and
215 a lack of a clutter banding indicated that the amplification was successful and that the
216 designed primer had specificity.
217
218 ##The correlation between the TIM-4 gene SNP and CHD
219 To explore the association between TIM-4 SNPs rs6882076 and rs7700944 with the
220 genetic susceptibility of CHD, the genotype and allele distribution of the TIM-4 gene
221 rs7700944 and rs6882076 in the healthy control group and CHD group were determined
222 with the Hardy-Weinberg equilibrium test (P>0.05). There were significant differences
223 in rs7700944 locus genotype and allele distribution frequencies between the healthy
224 control group and CHD group (P<0.05, Table 2). However, there was no significant
225 difference in rs6882076 locus genotype and allele frequency between the two groups
226 (P>0.05, Table 3). Therefore, the TIM-4 gene rs7700944 locus is related to CHD, while
227 the rs7700944AA homozygous type may be a protective factor against CHD.
228
229 ##Relationship between the different genotypes of the TIM-4 gene rs7700944 and
230 serum sTIM-4 levels and coronary scores in patients with CHD
231 Since the TIM-4 gene rs7700944 locus is related to CHD, we found that the level of
232 sTIM-4 in homologous serum of rs7700944 in patients with CHD was significantly
233 higher than that in patients with the GA genotype (P<0.001, Figure 6A). Further
234 indicating that the homozygous TIM-4 gene rs7700944 AA may be a protective gene
235 against CHD. In terms of coronary score, the AA genotype had a higher score than did
236 the GG type (P<0.05, Figure 6B). This indicates that serum sTIM-4 will probably be an
237 independent predictor of the severity of CHD in patients with homozygous rs770094
238 AA CHD.
239
240 #Discussion
241
242 With the rapid development of modern medical technology and the invention of new
243 gene detection technologies, a number of genes closely related to CHD have been
244 discovered (15). Meta-analyses indicate that the adiponectin gene and GP78 gene
245 polymorphisms are associated with CHD (16,17). The research on the association of
246 genes and CHD has proliferated enormously. These studies provide a new path toward
247 further understanding the pathogenesis and mechanism of CHD as well as its treatment
248 and early intervention.
249
250 CHD is characterized by lipid accumulation and immune inflammatory response (18).
251 The clearance of apoptotic cells plays an important role in maintaining immune
252 homeostasis (19,20). In the early formation of atherosclerotic plaques, clearance of
253 apoptotic cells can inhibit the inflammatory response and limit the increase in plaque
254 (21). In the later stage of atherosclerotic plaque formation, the clearance of apoptotic
255 cells induces an inflammatory response, which in turn promotes the formation of
256 atherosclerosis (22). Apoptotic cells can be identified by different signals. The exposed
257 cell membrane phosphatidylserine (PS) can release the “eat me” signal, which promotes
258 the uptake of PS receptor-derived phagocytic cells and the clearing of apoptotic cells
259 (23). The TIM protein is a type I transmembrane glycoprotein expressed in a variety of
260 immune cells that can recognize cells exposed to PS, regulate synergistic stimulation,
261 and inhibit signals (24).
262
263 The TIM-4 gene is located on human chromosome 5. TIM-4 is a natural ligand for TIM-
264 1, which is mainly expressed on the surface of cell membranes of APCs such as mature
265 dendritic cells and macrophages. TIM-4 acts in conjunction with its receptor TIM-1,
266 and they promote the differentiation and proliferation of T helper 2 (Th2) cells and can
267 also regulate the immune response (25-27). In addition, TIM-4 is a receptor for PS,
268 which promotes the phagocytosis of phagocytic apoptotic cells by recognizing the
269 binding of PS to cells. If TIM-4 is blocked, it can aggravate the progression of
270 atherosclerosis (7).
271
272 In this study, AS-qRT-PCR was used for genotyping. This type of SNP detection method
273 has many advantages. First, it is suitable for automatic operation; is simple, fast, and
274 low cost; and requires few special reagents. Second, the impure samples can also be
275 reliably analyzed, with the data analysis being simple and easy to automate. Compared
276 with AS-PCR, AS-qRT-PCR primer design does not require the introduction of a
277 mismatch, which increases the success rate of the experiment. It can also reduce the gel
278 electrophoresis step, the experimental steps, and pollution, thus saving time. Compared
279 to TaqMan probe technology, AS-qRT-PCR can reduce costs and the cycle of synthetic
280 probes, and it is also cheaper compared to SNP gold standard sequencing. The results
281 of AS-qRT-PCR using qPCR technology are accurate and reliable, and it is an ideal
282 experimental method for detecting a large sample of SNPs with few sites.
283
284 A SNP, as its name suggests, is a substitution of a single nucleotide that occurs at a
285 specific position in the genome, where each variation is present to some appreciable
286 degree within a population. More than 84 million SNPs have been found across humans
287 from multiple populations. A typical genome differs from the reference human genome
288 at 4 to 5 million sites, most of which (more than 99.9%) consist of SNPs and indels. By
289 reviewing the international literature on disease-related SNPs, we selected rs6882076
290 [minor allele frequency (MAF) =0.244] in the functional promoter region and
291 rs7700944 (MAF =0.2746) in the fifth intron variant. In the study, the 2 SNPs were
292 labeled as SNPs, with each representing a linkage equilibrium block.
293
294 This study found that patients with CHD had reduced levels of sTIM-4 and that
295 individuals with rs7700944 AA genotype were less susceptible to CHD. Moreover,
296 patients with CHD and the rs7700944 AA genotype had higher serum sTIM-4 levels,
297 whereas patients with CHD and the rs7700944 AA genotype had higher coronary scores,
298 indicating that their genotype had no correlation with the severity of CHD. The
299 rs7700944 locus is located in the G>A mutation in intron 5 of the TIM-4 gene. However,
300 the manner in which the TIM-4 gene functions in CHD, the pathway involved in
301 regulating CHD, and whether serum sTIM-4 levels can be used for the early diagnosis
302 and prognosis of CHD also warrant further investigation. These will be the key
303 scientific issues under focus in our follow-up studies.
304
305 Acknowledgments
306 We would like to thank the supporting from the Collaborative Innovation Center of
307 Technology and Equipment for Biological Diagnosis and Therapy in the Universities
308 of Shandong as well as Professor Fang Zheng (Shandong University) for data analysis.
309 Funding: This work was supported by the National Natural Science Foundation of
310 China (Nos. 81971480 and 81670520) and the Joint Fund Project of the Natural Science
311 Foundation of Shandong Province (No. ZR2019LZL013).
312
313 Footnote
314 Conflicts of Interest: The authors have no conflicts of interest to declare.
315
316 Ethical Statement: The authors are accountable for all aspects of the work in ensuring
317 that questions related to the accuracy or integrity of any part of the work are
318 appropriately investigated and resolved.
319
320 Highlight box
321
322 Key findings
323 l TIM-4, acting as a receptor for PS, promotes the phagocytosis of apoptotic
324 cells. However, the relationship between TIM-4 gene polymorphisms and
325 CHD remains unclear at present.
326 What is known and what is new?
327 l Our results showed that the TIM-4 gene rs7700944 is significantly correlated
328 with CHD, with the AA genotype being less sensitive to CHD.
329 l The level of sTIM-4 in the homologous serum of rs7700944 in patients with
330 CHD was significantly higher than that in patients with the GA genotype.
331 What is the implication, and what should change now?
332 l Our results indicated that the homozygous TIM-4 gene rs7700944 AA may
333 exert a protective effect against CHD.
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401 (English Language Editor: J. Gray)
402
403 Figure 1 sTIM-4 levels in serum from CHD patients and healthy controls.
404 ***, P< 0.001.
405

406
407 Figure 2 Heterozygous genotype with the same CT value, the melting curve, and
408 temperature. CT, cycle threshold.
409
410 Figure 3 Homozygous genotype with different CT values, the melting curve, and
411 temperature. CT, cycle threshold.
412
413

414
415 Figure 4 Electrophoresis and genotyping of rs7700944. (A) Electrophoresis of PCR
416 products for sequencing and genotyping at rs770094. (B) Genotype GG at rs7700944.
417 (C) Genotype GA at rs7700944. (D) Genotype AA at rs7700944. PCR, polymerase
418 chain reaction.
419
420 Figure 5 Electrophoresis and genotyping of rs6882076. (A) Electrophoresis of PCR
421 products for sequencing at rs6882076. (B) Electrophoresis of PCR products for
422 genotyping at rs6882076. (C) Genotype CT at rs6882076. (D) Genotype CC at
423 rs6882076. (E) Genotype TT at rs6882076. PCR, polymerase chain reaction.
424
425

426
427 Figure 6 Association of serum sTIM-4 and coronary scores with TIM-4 genotype at
428 rs7700944 in CHD patients (A) Association of serum sTIM-4 and coronary scores
429 with the TIM-4 genotype at rs7700944 in patients with CHD. (B) Association of
430 coronary scores with the TIM-4 genotype at rs7700944 in patients with CHD. *,P<
431 0.05 ; ****,P< 0.001; ns, not significant. sTIM-4, soluble TIM-4; TIM-4, T-cell
432 immunoglobulin and mucin domain containing 4; CHD, coronary atherosclerotic
433 heart disease.
434 Table 1 Characteristics of healthy controls and patients with CHD.
Variable Control (N=500) % CHD (N=499) % P value
Age,years 56.17±9.38 60.85±10.20 <0.001
Sex
Male 282 56.4 340 68.18
0.002
Female 218 43.6 159 31.82
Smoke
Yes 89 17.8 211 42.31
<0.001
No 411 82.2 288 57.69
Alcohol use
Yes 80 16.01 183 36.71
<0.001
No 420 83.99 316 63.29
TG(mmol/L) 1.25±0.60 1.76±1.16 <0.001
TC(mmol/L) 4.65±0.82 4.53±1.1 0.0603
HDL-C(mmol/L) 1.27±0.31 1.12±0.31 <0.001
LDL-C(mmol/L) 2.91±0.77 2.80±1.01 0.0885
435 CHD, coronary atherosclerotic heart disease; TG, triglyceride; TC, total cholesterol;
436 HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein
437 cholesterol.
438
439
440
441 Table 2 Genotype and allele frequencies between patients with CHD and healthy
442 controls at rs7700944 of the TIM-4 gene
Group Case Genotype，N（%） Allele，N（%）
GG GA AA G A
CHD 499 309(61.9) 167(33.5) 23(4.7) 784(78.6) 214(21.4)
Cntrool 500 294(58.8) 158(31.6) 48(9.6) 746(74.6) 254(25.4)
χ2 9.344 4.527
Ρ 0.009 0.033
443 CHD, coronary atherosclerotic heart disease; TIM-4, T-cell immunoglobulin and mucin
444 domain containing 4.
445 Table 3 Genotype and allele frequencies between the patients with CHD and healthy
446 controls at rs6882076 of the TIM-4 gene
Group Case Genotype，N（%） Allele，N（%）
CC CT TT C T
CHD 499 281(56.4) 184(36.8) 34(6.8) 747(74.8) 251(25.2)
Cntrool 412 225(54.6) 149(36.2) 38(9.2) 599(72.7) 225(27.3)
χ2 1.892 1.059
Ρ 0.388 0.303

447 CHD, coronary atherosclerotic heart disease; TIM-4, T-cell immunoglobulin and mucin
448 domain containing 4.
449
450
451
452 Materials Design Analysis Reporting (MDAR)
453 Checklist for Authors
454
455 The MDAR framework establishes a minimum set of requirements in transparent reporting applicable to studies
456 in the life sciences (see Statement of Task: doi:10.31222/osf.io/9sm4x.). The MDAR checklist is a tool for authors,
457 editors and others seeking to adopt the MDAR framework for transparent reporting in manuscripts and other
458 outputs. Please refer to the MDAR Elaboration Document for additional context for the MDAR framework.
459
460 Materials
461
Antibodies Yes (indicate where provided: section/paragraph) n/a

For commercial reagents, provide supplier name, √

catalogue number and RRID, if available.

Cell materials Yes (indicate where provided: section/paragraph) n/a

Cell lines: Provide species information, strain. √

Provide accession number in repository OR supplier
name, catalog number, clone number, OR RRID

Primary cultures: Provide species, strain, sex of √

origin, genetic modification status.

Experimental animals Yes (indicate where provided: section/paragraph) n/a

Laboratory animals: Provide species, strain, sex, age, √
genetic modification status. Provide accession number in
repository OR supplier name, catalog number, clone
number, OR RRID
Animal observed in or captured from the field: √
Provide species, sex and age where possible
Model organisms: Provide Accession number in √
repository (where relevant) OR RRID
 Plants and microbes Yes (indicate where provided: section/paragraph) n/a
Plants: provide species and strain, unique accession √
number if available, and source (including location for
collected wild specimens)
Microbes: provide species and strain, unique √
accession number if available, and source

Human research participants Yes (indicate where provided: section/paragraph) n/a

Identify authority granting ethics approval (IRB or Line83-85

equivalent committee(s), provide reference number for
approval.

Provide statement confirming informed consent obtained √

from study participants.
Report on age and sex for all study participants. √
462
463 Design
464
Study protocol Yes (indicate where provided: section/paragraph) n/a
For clinical trials, provide the trial registration number OR √
cite DOI in manuscript.

Laboratory protocol Yes (indicate where provided: section/paragraph) n/a

Provide DOI or other citation details if detailed step-by- √
step protocols are available.

Experimental study design (statistics details) Yes (indicate where provided: section/paragraph) n/a

State whether and how the following have been done, or if

they were not carried out.
Sample size determination Line82-83
Randomisation √
Blinding √
Inclusion/exclusion criteria √

Sample definition and in-laboratory replication Yes (indicate where provided: section/paragraph) n/a

State number of times the experiment was replicated in √

laboratory
Define whether data describe technical or biological √
replicates

Ethics Yes (indicate where provided: section/paragraph) n/a

Studies involving human participants: State details of Line83-85
authority granting ethics approval (IRB or equivalent No.ECSBMSSDU2019-1-010
committee(s), provide reference number for approval.
Studies involving experimental animals: State details of √
authority granting ethics approval (IRB or equivalent
committee(s), provide reference number for approval.
Studies involving specimen and field samples: State if √
relevant permits obtained, provide details of authority
approving study; if none were required, explain why.
465
 Dual Use Research of Concern (DURC) Yes (indicate where provided: section/paragraph) n/a
If study is subject to dual use research of concern, state √
the authority granting approval and reference number for
the regulatory approval

466
467 Analysis
468
Attrition Yes (indicate where provided: section/paragraph) n/a
State if sample or data point from the analysis is excluded, √
and whether the criteria for exclusion were determined
and specified in advance.

Statistics Yes (indicate where provided: section/paragraph) n/a

Describe statistical tests used and justify choice of tests. Line132-136

Data Availability Yes (indicate where provided: section/paragraph) n/a

State whether newly created datasets are available, √

including protocols for access or restriction on access.
If data are publicly available, provide accession number in √
repository or DOI or URL.
If publicly available data are reused, provide accession √
number in repository or DOI or URL, where possible.

Code Availability Yes (indicate where provided: section/paragraph) n/a

For all newly generated code and software essential for
replicating the main findings of the study:
State whether the code or software is available. √
If code is publicly available, provide accession number in √
repository, or DOI or URL.
469
470 Reporting
471
Adherence to community standards Yes (indicate where provided: section/paragraph) n/a
MDAR framework recommends adoption of discipline-
specific guidelines, established and endorsed through
community initiatives. Journals have their own policy
about requiring specific guidelines and recommendations
to complement MDAR.

State if relevant guidelines (eg., ICMJE, MIBBI, ARRIVE) √

have been followed, and whether a checklist (eg.,
CONSORT, PRISMA, ARRIVE) is provided with the
manuscript.

472
Please leave this space alone as it will be supplemented by the editorial office when needed.

473