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Disease Markers
Volume 2018, Article ID 1064380, 5 pages
https://doi.org/10.1155/2018/1064380
Research Article

Association of Thrombomodulin Gene C1418T Polymorphism with Susceptibility to Kawasaki Disease in Chinese Children

1Department of Pediatrics, The Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China
2Central Laboratory, The Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China

Correspondence should be addressed to Zuocheng Yang; moc.621@rcz_gnay

Received 3 March 2018; Accepted 3 May 2018; Published 12 June 2018

Academic Editor: Michael Hawkes

Copyright © 2018 Yapeng Lu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Kawasaki disease (KD) is an acute systemic vasculitis that predominantly affects children and can result in coronary artery lesions (CALs). Thrombomodulin (TM) is a critical cofactor in the protein C anticoagulant system. The TM C1418T (rs1042579) polymorphism is associated with a high risk of cardiac-cerebral vascular diseases. But the association of the TM C1418T polymorphism with susceptibility to KD, CAL formation, and intravenous immunoglobulin (IVIG) resistance is still unclear. In our study, we examined the TM C1418T polymorphism in 122 children with KD and 126 healthy children and revealed the correlation between the TM C1418T polymorphism and KD, CAL formation, and IVIG resistance.

1. Introduction

Kawasaki disease (KD) also known as mucocutaneous lymph node syndrome (MCLS) was first described by Tomisaku Kawasaki in 1967. It is characterized by prolonged fever, diffuse mucosal inflammation, indurative edema of the hands and feet, a polymorphous skin rash, and nonsuppurative lymphadenopathy. KD is a multisystem vasculitis that can result in coronary artery lesions (CALs), which are known to predominantly affect young children (84%~86% of cases occur in children between 6 months and 5 years of age) with a male predominance (approximately 1.5~1.8 times higher than females) [1]. The disease is self-limited; patients with KD mostly have a good prognosis with active treatment [2]. Coronary artery involvement is the most common complication of KD and may cause significant coronary stenosis, which results in coronary artery aneurysm and occlusion, leading to myocardial ischemia and infarction lesions. In recent years, the incidence rate of KD has increased [3]. A recent study on the epidemiology of KD described that the disease has replaced rheumatic heart disease as the most common cause of acquired heart disease in children in developed countries and in some developing countries [4]. KD is considered to be a potential risk factor for ischemic heart disease in adults and sudden cardiac death in young adults [5].

The etiopathogenesis of KD has not yet been clearly identified. It is speculated that KD is the result of the interaction among infection, immune dysfunction, and genetic susceptibility. There are significant differences in the morbidity among different racial groups; for example, Asian children are significantly more susceptible [6]. The incidence in siblings of KD patients and in offspring of parents with a history of KD is higher than that of the general population, which indicates that genetic susceptibility may play an important role in the pathogenesis of this disease [7]. In addition, recent research demonstrates that some single-nucleotide polymorphism (SNP) loci of genes regulating immune, inflammation, and blood coagulation, such as the tumor necrosis factor (TNF) gene, interleukin-related genes, and platelet endothelial cell adhesion molecule-1 (PECAM-1), have a close relationship with KD [810].

Thrombomodulin is considered a marker of vascular endothelial injury [11]. TM gene polymorphisms may be associated with myocardial infarction and cerebral infarction [12, 13], but it is unclear whether there is an association between the pathogenesis of KD and TM gene polymorphisms.

The objective of this study is to verify the hypothesis that the TM C1418T polymorphism may play a role in the risk assessment of KD. To the best of our knowledge, this is the first study that investigates the role of the TM C1418T polymorphism in KD.

2. Materials and Methods

2.1. Study Subjects

A total of 122 patients with KD were enrolled from January 2012 to December 2016 in the Third Xiangya Hospital, Changsha, China. All patients met the diagnostic criteria of the Japanese Kawasaki Disease Research Committee [14]. Patients were Han Chinese individuals 2 months to 9 years and 6 months of age. There were 82 males and 40 females with an average age at onset of 29.4 ± 22.2 months. Before hospital admittance, all patients had not received IVIG or aspirin treatment. To further investigate the relationship between CAL formation and TM gene polymorphisms, the KD group was divided into KD with coronary artery lesions (KD-CAL) and KD without coronary artery lesions (KD-WO) subgroups. All patients with KD received a series of imaging examinations, including pulse Doppler, two-dimensional and color flow echocardiogram, at least 3 times within 8 weeks from the onset of the illness. Echocardiographic follow-up was performed every 3 to 6 months in the first year for KD patients with abnormal coronary arteries and then once annually until the affected coronary arteries normalized. We used two-dimensional echocardiography to visualize the diameter of the left and right coronary arteries on the parasternal short-axis view of the aorta. In the echocardiogram, CAL is defined as the internal lumen diameter, of either the left or right coronary artery, of >3 mm in children <5 years of age and >4 mm in children ≥5 years of age; CAL can be also defined as the internal diameter of a segment that is at least 1.5 times that of an adjacent vessel or the coronary lumen is clearly irregular [15]. To investigate the relationship between intravenous immunoglobulin (IVIG) resistance and TM gene polymorphisms, we distinguished 17 patients resistant to IVIG as the IVIG-resistant group and 93 patients sensitive to IVIG as the IVIG-sensitive group. IVIG resistance was defined as a return of fever (rectal ) associated with one or more of the initial symptoms that led to the diagnosis of KD within 2~7 days or even 2 weeks after the initial IVIG treatment. A total of 126 healthy controls were recruited from January 2012 to December 2016 in the Third Xiangya Hospital, Changsha, China. Healthy controls were 5 to 14 years of age and did not have any previous history of KD, infectious diseases, cardiovascular diseases, or rheumatic diseases. There is no statistically significant gender distribution difference between the KD patients and healthy controls ().

We collected blood samples from KD patients and the controls in disposable blood collection tubes (containing anticoagulant EDTA-Na2). Blood samples were immediately transferred to cryopreservation tubes and stored at −80°C. The study protocol was approved by the Ethics Review Committee of Medical Research Institute and Institutional Review Boards of the Medical Research Institute at the Third Xiangya Hospital.

2.2. DNA Extraction

Genomic DNA was isolated from peripheral blood leukocytes using a Wizard genomic DNA purification kit according to the manufacturer’s instructions (Promega, Madison, WI, USA). Extracted DNA was detected by a NanoDrop ND-2000C Spectrophotometer (Thermo, USA).

2.3. Genotyping

The detection of genotypes of the TM C1418T polymorphism was tested with the polymerase chain reaction-sequence-based typing (PCR-SBT) method. The TM gene was amplified by polymerase chain reaction (PCR) using primers designed by the authors (forward: 5-GCTACATCCTGGACGACGGTTTC-3 and reverse: 5-GGCAGAGGAGCGCCAAAAGC-3) and a Thermal Cycler 9700 (Applied Biosystems, Foster City, CA, USA). PCR reactions were carried out in a total volume of 20 μL containing 1.6 μL of DNA, 10 μL of 2x Taq Master Mix (CWBio, China), 0.8 μL of forward primer (Sangon Biotech, China), 0.8 μL of reverse primer (Sangon Biotech, China), and ddH2O (added to a final volume of 20 μL). The following thermal cycling conditions were used: denaturing at 94°C for 5 minutes, followed by 35 cycles of denaturing at 94°C for 30 seconds, annealing at 66°C for 30 seconds, and extension at 72°C for 30 seconds, and then an extension at 72°C for 10 minutes. The amplification products were sent to a sequencing company (BioSune, China) for genetic sequencing. The sequences were read with the help of Chromas software.

2.4. Statistical Analysis

All statistical analyses were performed using SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA). The test with 1 degree of freedom was used to perform the Hardy-Weinberg equilibrium (HWE). The differences in genotype and allele frequencies between the two groups were evaluated using a test. The odds ratio (OR), along with its 95% confidence interval (95% CI), were estimated for associations between risk alleles and genotypes of KD. Two-sided were considered statistically significant.

3. Results

3.1. Association of TM Gene C1418T Polymorphism with Susceptibility to KD

The 324 bp amplification products were separated using gel electrophoresis (Figure 1). The PCR sequence-based typing results are shown in Figure 2. The genotypes of the single-nucleotide polymorphism were in line with the HWE for cases and controls (, 2.46, ). As shown in Tables 13, the distribution of genotypes (CT, TT) in the KD group was different from that in the control group (, ); carriers of allele T had a 1.852 times higher risk of KD than that of noncarriers (, , , 95% ).

Figure 1: Agarose electrophoresis charts of TM gene PCR products. M: DNA marker. Lanes 1–3: TM gene PCR products (amplification length: 324 bp).
Figure 2: The genotype test at locus C1418T in the TM gene. (a) CT genotype; (b) CC genotype; (c) TT genotype.
Table 1: Frequencies of TM genotypes in the study subjects.
Table 2: Frequencies of TM alleles in the study subjects.
Table 3: Genotype and allele frequencies of the TM Gene in controls and patients with KD.
3.2. Association of TM Gene C1418T Polymorphism with CAL Formation

As shown in Table 1, there were no significant differences in frequencies of genotypes (CC, CT, and TT) and alleles (C, T) between the KD-CAL group and the KD-WO group ().

3.3. Association of TM Gene C1418T Polymorphism with IVIG Resistance

As shown in Table 1, there were no significant differences in frequencies of genotypes (CC, CT, and TT) and alleles (C, T) between the IVIG-resistant group and the IVIG-sensitive group ().

4. Discussion

The TM gene is located on chromosome 20p11.2 and contains a single exon and no introns. TM is expressed primarily on the luminal surface of vascular endothelial cells and consists of 557 amino acids (aa): an N-terminal lectin-like module (aa 1–154), a hydrophobic region (aa 155–222), six epidermal growth factor- (EGF-) like modules (aa 223–462), a serine- and threonine-rich region (aa 463–497), a single transmembrane segment (aa 498–521), and a short cytoplasmic tail (aa 522–557). There are 6 EGF-like repeats at the EGF-like domain of the extracellular region, and the last 3 repeats are functionally important for protein C activation and thrombin binding [16]. The enzymatic cleavage from the cell surface produces soluble thrombomodulin (sTM) in the plasma. TM has at least three major anticoagulant characteristics: (1) it catalyzes thrombin activation of protein C; (2) it alters thrombin substrate specificity, which leads to inhibition of thrombin-mediated clotting, platelet activation, and procoagulant factors (V, VIII, XI, and XIII); and (3) it plays a significant role in the inhibition of thrombin by antithrombin. The increasing level of sTM can be detected in various kinds of endothelial cell injuries, such as plasma, urine, and joint synovial fluid [17]. The normal vascular endothelium can secrete anticoagulants (TM, heparin, tissue plasminogen activator, etc.) to maintain local blood coagulation, fibrinolysis balance, and blood flow. We know that TM plays an important role in anticoagulation. In addition, TM can exert its anti-inflammatory effect via the activated protein C- (APC-) independent pathway and APC-dependent pathway [18]. The TM C1418T polymorphism, which encodes for the replacement of Ala455 by Val455 in the TM gene, has been well described in previous studies. This polymorphism is located in the coding region of the TM gene, which is responsible for thrombin binding and protein C activation, signifying its potential role in regulating thrombomodulin function [16].

The TM C1418T polymorphism has been widely found to be associated with cardiac-cerebral vascular diseases. Wang and Dong [19] conducted a meta-analysis study to uncover the association between two SNPs in the TM gene, −33G/A and Ala455Val (C1418T), and coronary artery disease (CAD), and they reported a significant association of the two loci of the gene polymorphism with CAD. Lobato et al. [20] found that allele T of the locus C1418T in the TM gene is independently associated with increased long-term mortality risk following coronary artery bypass grafting (CABG) and significantly improved the classification ability of traditional postoperative mortality prediction models. A study from north India showed that the TM C1418T polymorphism is an independent predictor of acute myocardial infarction (AMI) [21].

According to previous studies, there is a close relationship between the development of KD and endothelial cell injury, a hypercoagulable state, and thrombosis tendency [22]. Thus, TM gene polymorphisms may be associated with KD, but there has been no published research in this field.

The present study results illustrate that there were significant differences in genotype frequencies of CC, CT, and TT between the KD group and the control group. In addition, carriers with genotype CT had a 2.356 times higher risk of KD than that of noncarriers, and carriers with allele T had a 1.852 times higher risk of KD than that of noncarriers. However, there were no significant differences in frequencies of genotypes (CC, CT, and TT) or alleles (C, T) between the KD-CAL and KD-WO groups and IVIG-resistant and IVIG-sensitive groups. These findings suggest that the genotype CT and allele T may be susceptible factors to KD because of the significant correlation between the TM C1418T polymorphism and KD. However, the TM C1418T polymorphism was not found to be associated with CAL formation or IVIG resistance. There are several possible reasons for this finding: the sample size was not sufficient to insure the accuracy of the results; C1418T may not be the responsible locus for the association between the TM gene polymorphism, CAL formation, and IVIG resistance; or there is indeed no specific relationship between the TM gene polymorphism, CAL formation, and IVIG resistance. Hence, further larger studies with more information could verify our findings.

In conclusion, our study illuminated that the TM C1418T polymorphism was significantly associated with the risk of KD.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that there are no competing interests regarding the publication of this article.

Authors’ Contributions

Yapeng Lu and Rui Liu contributed equally to this work.

Acknowledgments

This work was supported by the New Xiangya Talent Project of the Third Xiangya Hospital of the Central South University (Grant no. JY20150312).

References

  1. D. Yim, N. Curtis, M. Cheung, and D. Burgner, “Update on Kawasaki disease: epidemiology, aetiology and pathogenesis,” Journal of Pediatrics and Child Health, vol. 49, no. 9, pp. 704–708, 2013. View at Publisher · View at Google Scholar · View at Scopus
  2. K. Y. Kim and D. S. Kim, “Recent advances in Kawasaki disease,” Yonsei Medical Journal, vol. 57, no. 1, pp. 15–21, 2016. View at Publisher · View at Google Scholar · View at Scopus
  3. S. Singh, P. Vignesh, and D. Burgner, “The epidemiology of Kawasaki disease: a global update,” Archives of Disease in Childhood, vol. 100, no. 11, pp. 1084–1088, 2015. View at Publisher · View at Google Scholar · View at Scopus
  4. S. Singh, S. Bhattad, A. Gupta, D. Suri, A. Rawat, and M. Rohit, “Mortality in children with Kawasaki disease: 20 years of experience from a tertiary care centre in North India,” Clinical and Experimental Rheumatology, vol. 34, Supplement 97, no. 3, pp. S129–S133, 2016. View at Google Scholar
  5. H. Kato, T. Sugimura, T. Akagi et al., “Long-term consequences of Kawasaki disease. A 10- to 21-year follow-up study of 594 patients,” Circulation, vol. 94, no. 6, pp. 1379–1385, 1996. View at Publisher · View at Google Scholar · View at Scopus
  6. N. Makino, Y. Nakamura, M. Yashiro et al., “Descriptive epidemiology of Kawasaki disease in Japan, 2011–2012: from the results of the 22nd nationwide survey,” Journal of Epidemiology, vol. 25, no. 3, pp. 239–245, 2015. View at Publisher · View at Google Scholar · View at Scopus
  7. R. Uehara, M. Yashiro, Y. Nakamura, and H. Yanagawa, “Parents with a history of Kawasaki disease whose child also had the same disease,” Pediatrics International, vol. 53, no. 4, pp. 511–514, 2011. View at Publisher · View at Google Scholar · View at Scopus
  8. S. Arj-Ong, A. Thakkinstian, M. McEvoy, and J. Attia, “A systematic review and meta-analysis of tumor necrosis factor α-308 polymorphism and Kawasaki disease,” Pediatrics International, vol. 52, no. 4, pp. 527–532, 2010. View at Publisher · View at Google Scholar · View at Scopus
  9. Z. Li, D. Han, J. Jiang, J. Chen, L. Tian, and Z. Yang, “Association of PECAM-1 gene polymorphisms with Kawasaki disease in Chinese children,” Disease Markers, vol. 2017, Article ID 2960502, 6 pages, 2017. View at Publisher · View at Google Scholar · View at Scopus
  10. K. P. Weng, K. S. Hsieh, Y. T. Hwang et al., “IL-10 polymorphisms are associated with coronary artery lesions in acute stage of Kawasaki disease,” Circulation Journal, vol. 74, no. 5, pp. 983–989, 2010. View at Publisher · View at Google Scholar · View at Scopus
  11. C. Mikacenic, W. O. Hahn, B. L. Price et al., “Biomarkers of endothelial activation are associated with poor outcome in critical illness,” PLoS One, vol. 10, no. 10, article e141251, 2015. View at Publisher · View at Google Scholar · View at Scopus
  12. A. A. Azme, D. K. Shome, A. H. Salem, S. A. Fadhli, R. A. Bannay, and A. Jaradat, “Thrombomodulin gene proximal promoter polymorphisms in premature acute coronary syndrome patients in Bahrain,” Blood Coagulation & Fibrinolysis, vol. 26, no. 8, pp. 919–924, 2015. View at Publisher · View at Google Scholar · View at Scopus
  13. J. W. Cole, S. C. Roberts, M. Gallagher et al., “Thrombomodulin Ala455Val polymorphism and the risk of cerebral infarction in a biracial population: the stroke prevention in young women study,” BMC Neurology, vol. 4, no. 1, p. 21, 2004. View at Publisher · View at Google Scholar · View at Scopus
  14. M. Ayusawa, T. Sonobe, S. Uemura et al., “Revision of diagnostic guidelines for Kawasaki disease (the 5th revised edition),” Pediatrics International, vol. 47, no. 2, pp. 232–234, 2005. View at Publisher · View at Google Scholar · View at Scopus
  15. “Guidelines for diagnosis and management of cardiovascular sequelae in Kawasaki disease,” Pediatrics International, vol. 47, no. 6, pp. 711–732, 2005. View at Publisher · View at Google Scholar · View at Scopus
  16. G. Anastasiou, A. Gialeraki, E. Merkouri, M. Politou, and A. Travlou, “Thrombomodulin as a regulator of the anticoagulant pathway: implication in the development of thrombosis,” Blood Coagulation & Fibrinolysis, vol. 23, no. 1, pp. 1–10, 2012. View at Publisher · View at Google Scholar · View at Scopus
  17. M. H. Strijbos, C. Rao, P. I. Schmitz et al., “Correlation between circulating endothelial cell counts and plasma thrombomodulin levels as markers for endothelial damage,” Thrombosis and Haemostasis, vol. 100, no. 4, pp. 642–647, 2008. View at Publisher · View at Google Scholar · View at Scopus
  18. R. Carnemolla, K. R. Patel, S. Zaitsev, D. B. Cines, C. T. Esmon, and V. R. Muzykantov, “Quantitative analysis of thrombomodulin-mediated conversion of protein C to APC: translation from in vitro to in vivo,” Journal of Immunological Methods, vol. 384, no. 1-2, pp. 21–24, 2012. View at Publisher · View at Google Scholar · View at Scopus
  19. H. Wang and P. Dong, “Thrombomodulin −33G/A and Ala455Val polymorphisms are associated with the risk of coronary artery disease: a meta-analysis including 12584 patients,” Coronary Artery Disease, vol. 26, no. 1, pp. 72–77, 2015. View at Publisher · View at Google Scholar · View at Scopus
  20. R. L. Lobato, W. D. White, J. P. Mathew et al., “Thrombomodulin gene variants are associated with increased mortality after coronary artery bypass surgery in replicated analyses,” Circulation, vol. 124, Supplement 1, no. 11, pp. S143–S148, 2011. View at Publisher · View at Google Scholar · View at Scopus
  21. R. Dogra, R. Das, J. Ahluwalia, R. M. Kumar, and K. K. Talwar, “Association of thrombomodulin gene polymorphisms and plasma thrombomodulin levels with acute myocardial infarction in north Indian patients,” Clinical and Applied Thrombosis/Hemostasis, vol. 19, no. 6, pp. 637–643, 2013. View at Publisher · View at Google Scholar · View at Scopus
  22. T. Yahata, C. Suzuki, A. Yoshioka, A. Hamaoka, and K. Ikeda, “Platelet activation dynamics evaluated using platelet-derived microparticles in Kawasaki disease,” Circulation Journal, vol. 78, no. 1, pp. 188–193, 2014. View at Publisher · View at Google Scholar · View at Scopus