Whole-exome sequencing analysis identifies novel variants associated with Kawasaki disease susceptibility

DOI: https://doi.org/10.21203/rs.3.rs-2272385/v1

Abstract

Background: Kawasaki disease (KD) is an acute pediatric vasculitis affecting genetically susceptible infants and children. Although the pathogenesis of KD remains unclear, growing evidence links genetic susceptibility to the disease. To explore the genes associated with susceptibility in KD, we conducted whole-exome sequencing of KD children from Yunnan province, China.

Methods: We retrospectively evaluated the data from 93 KD patients and 91 non-KD controls who underwent whole-exome sequencing.

Results: In this study, we successfully collected and identified relationships between two significant rare variant genes (MYH14 and RBP3) and Kawasaki disease through the genotype/allele frequency analysis (odds ratio [OR], 8.3945 to 13.1963; p-value, 0.0025 to 0.0346). The eight of 20 KD patients all have heterogeneous variants in Chr19: 50281727 (G/A), Chr19: 50223086 (G/A), Chr19: 50280044 (T/G), Chr19: 50301707 (C/A), Chr19: 50301790 (C/T), Chr19: 50293670 (C/T), Chr19: 50292282 (C/T), Chr19: 50244260 (C/T), and the remaining twelve cases had heterogeneous variants in Chr10: 47351134 (G/A), associated with retinitis pigmentosa, which may be associated to one clinal manifestation of KD.

Conclusion: This study suggested that two genes MYH14 and RBP3 may be associated with KD susceptibility in the population from Yunnan province.

Introduction

Kawasaki disease (KD; OMIM611775) is an acute, self-limiting systemic vasculitis syndrome with the main clinical manifestations of fever, oral mucosal changes, rash, cervical lymphadenopathy, bulbar conjunctival hyperemia, and extremity changes, known as mucocutaneous lymph node syndrome (MCLS) [1, 2]. It was first described by Japanese pediatrician Tomisaku Kawasaki and particularly affects children under five years of age. With an almost worldwide increase in incidence, KD becomes now the leading cause of acquired heart disease in children, as it may cause coronary artery lesions in 15–25% of untreated patients or in 5–10% of patients treated with intravenous immunoglobulin [3]. The incidence of incomplete KD, which accounts for 15% and 47% of all KD cases [4], has also been reported to be increasing, posing a threat to the health of children’s coronary arteries [57].

Although the clinical symptoms of KD can be identified [8], the immunopathogenic mechanisms of this disease remain unclear. Common and rare genetic variants could form many complex traits with complex interactions [911]. Domestic and foreign studies have found that inflammation-related genes IL-18 and IL-1B [1, 12, 13], inositol 1, 4, 5-trisphosphate 3-kinase C (ITPKC) [14, 15], and other gene polymorphisms are associated with KD [16]. Meanwhile, the family aggregation of KD patients indicated that genetic factors play an important role in the occurrence of KD [1719]. However, the susceptibility loci obtained by the candidate gene method have been controversial because the results of various studies cannot lead to more accurate and consistent conclusions due to differences in race, environment, and sample content. Finding susceptibility genes associated with complex diseases at the genome-wide level is an effective approach to investigating polygenic diseases. Commonly used methods include genome-wide linkage studies, genome-wide association studies (GWAS), and whole-exome sequencing (WES) [20]. Most GWAS-derived Single nucleotide polymorphisms (SNPs) do not directly affect disease characteristics, but are an index marker linking disease-specific imbalances and pathogenic variants [16, 21, 22]. Therefore, it is necessary to use other methods to identify rare coding variants that affect KD susceptibility. WES is one of the efficient sequencing techniques to identify rare protein-coding variants. In this study, we determined to identify the KD-associated protein-coding variants through WES.

Materials And Methods

Patients and Samples

The diagnosis of KD was made according to the clinical criteria for KD. 93 patients with KD with a mean age of seven days to twelve years have signed a written informed consent before inclusion in the subsequent analyses, 18 of whom were ethnic minorities and the remainder were Han ethnic group. All KD cases and non-KD healthy control samples (n = 91) with unrelated kinship were obtained from the Kunming Children’s Hospital in China. This study was approved by the Ethical Review Board of Kunming Children’s Hospital, and informed consent was obtained from the parents of KD patients. Inclusion criteria for KD cases included patients with established KD, untreated, and no previous cancer or metastases.

Whole-exome Sequencing And Confirmation Via Sanger Sequencing

WES was conducted using genomic DNA samples obtained from 93 children with KD. The exome sequences were efficiently enriched from 1 µg genomic DNA extracted from the peripheral blood using Agilent liquid capture system (Agilent Sure Select Human All Exon V6 kit, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. Finally, Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA), with 150 bp pair fragments sequencing mode, was used for sequencing the genomic DNA for shotgun library construction. The overall genotyping success rate was 99.5%. Raw image files were processed using CASAVA v1.82 for base calling and generating raw data. Sanger sequencing of candidate variants was performed according to standard protocols.

Statistical Analysis

Variation frequencies were described as proportions, and SNP allele frequency comparisons between cases and controls were analyzed by Fisher's exact tests and odds ratios [19], and 95% confidence intervals (CIs) calculated by unconditional logistic regression were used to analyze the association between SNPs and KD susceptibility. Two-tailed p-value < 0.05 was considered statistically significant.

Results

Baseline Characteristics

The study included 93 KD patients, of which all patients had complete KD with a fever lasting longer than five days and four principal symptoms of KD such as conjunctivitis, skin rashes, and a decrease of fibrinogen, etc. without any drug treatments. Therefore, we performed an association analysis of 93 complete KD cases and 91 non-KD controls, whom all come from Yunnan province, southern China, and have no relative relationship with each other. The ratio of males to females in this study was approximately 2: 1 (slightly higher than the ratio of 1.5: 1 of KD patients generally, p-value < 0.05 according to Fisher's exact test).

Rare Variants Revealed Via Whole-exome Sequencing Associations With Kd

We identified common variants with a total of 349,054 SNPs (with minor allele frequency MAF < 0.01) from 93 KD cases and 91 controls, with a database involved 1000g2015aug_all, ExAC_ALL, ExAC_EAS, gnomAD_exome_ALL, gnomAD_exome_EAS, gnomAD_genome_ALL, gnomAD_genome_EAS. To filter out the susceptibility SNPs association to KD, missense mutation and macromutation including frameshift mutation, terminator mutation, and splice site mutation, where utilized with a Combined Annotation Dependent Depletion (CADD) [23] scores > 25. After filtering, 8413 protein-altering variants remained, among which 6466 were nonsynonymous SNV, 851 were frameshift, 394 were in splice-acceptor/donor sites, 687 were nonsense, and 15 were start lost. ORs were valued by R package ggplot2, which performed deleterious when ORs > 1. Through case-control association analysis of these 8413 variants, 11 variants (located in 3 genes) were identified with allele frequencies significantly different between the KD patients and non-KD controls (Table 1, Fig. 1).


Associations With Kd

We successfully collected three extremely rare variant genes (TTI1, MYH14, and RBP3) from 46 cases (Fig. 1 and Table 1), which showed nominal significances (ORs = 2.3177 to 13.1963; p = 0.0025 to 0.0346) (Table 1). And among these variants (possibly associated with KD), TTI1 was excluded as its allele frequency distribution performed too high at the same loci of control. The eight of 20 KD patients all have heterogeneous variants in Chr19: 50281727 (G/A), Chr19: 50223086 (G/A), Chr19: 50280044 (T/G), Chr19: 50301707 (C/A), Chr19: 50301790 (C/T), Chr19: 50293670 (C/T), Chr19: 50292282 (C/T), Chr19: 50244260 (C/T). These variants have been documented to be associated with deafness and schizophrenia in the HGMD study. And the remaining twelve cases had heterogeneous variants in Chr10: 47351134 (G/A), associated with retinitis pigmentosa.

Discussion

GWAS has identified some well-defined KD-associated loci and part of the genetic background successfully in recent studies, while it does not contribute significantly to exploring the pathogenesis of KD [24, 25]. Different from the GWAS, WES technology can explore global genetic mutations of many other complex diseases. It could discover rare mutations in the encoding sequence, which may cause its protein-coding variants that contribute to KD susceptibility. Recent studies have reported several rare protein encoding variants associated with KD by WES technology [20]. KD is a multisystem inflammatory process, presumably, the etiology is an excessive immune response to possible infection or environmental triggers in genetically susceptible individuals [26]. People with KD may be inherently prone to other diseases, especially children younger than five years. Researchers reported that the incidence of KD in Asia was higher than that in the United States and Europe [2630], and a higher incidence of males than females [2, 31]. Studies also have reported that the incidence between brothers or sisters was higher than that of usual people [17, 30].

In this study, we performed WES to identify rare protein-coding variants responsible for KD susceptibility. And comparative analysis with evidence obtained from previously reported articles, two rare coding variants genes MYH14 and RBP3 were discovered after filtering, which showed nominal significances (ORs = 8.3945; p = 0.0346) were associated with KD. There were six amino acid mutations distributed on seven chromosome positions of MYH14 (myosin Heavy Chain 14), and one mutation on Chr10: 47351134 of RBP3 (retinoid-binding protein 3). All the allele frequencies were lower than 0.0275%, which indicated both of them were rare variants of genes.

To date, sixteen RBP3 gene variants have been recorded in the HGMD database, including eleven missense variants, three-terminal variants, one frameshift variant, and one fragment deletion variant (Fig. 2). In 2015, Arno et al first described retinal dystrophy in children caused by homozygous nonsense RBP3 mutations, highlighting the requirement for IRBP in normal eye development and visual function [32]. Yokomizo et al [33] found that elevated expression of photoreceptor-secreted RBP3 may play a role in protection against the progression of diabetic retinopathy. While in this research, rs11204213 (c.2650G > A, p.V884M) has induced amino acid changes in Val884Met, a residue in the third of four tandem homology modules [28, 34, 35]. As compared with other SNPs, it recorded larger effects on ocular axial length, the growth of the eyeball, or the development of related phenotypes such as myopia from the missense RBP3 SNPs, Therefore, previous researchers just investigated that RBP3 was thought to be associated with retinal retinoid transport and corneal changes, however, it may be referred to KD in our research. For another rare variant MYH14, sixty gene variants have been recorded in the HGMD database, within 54 missense variants, three-terminal variants, and three frameshift variants. Among these gene variants, about eight missense variants were discovered in this study (Fig. 3), however, only gene variant A1798D (chr14: rs368219210, c.5393C > A, p.A1798D) has been published before. The only research reported that homozygous MYH14 mutations may cause perineal fistulas in Anorectal malformations, based on the genetic and computer analyses, and be related to normal cloaca development by NMHC IIC localization analysis [36]. Above all, RBP3 and MYH14 both have never been reported to be associated with KD susceptibility so far, however, one main clinical manifestation of bulbar conjunctival hyperemia in KD is appeared to be related to these rare variants in our search.

Conclusion

WES high-throughput was performed on the patients with KD to identify disease susceptibility genes in children from southwest China, and two rare protein-coding variants (RBP3 and MYH14) were identified in the genotype/allele frequency distribution (ORs, 8.3945 to 13.1963; p-value, 0.0346 to 0.0025), that may influence KD susceptibility. These results provide insights into novel candidate genes and genetic variants that may be involved in KD and related KD complications. Further association studies with expanded KD samples from southwestern China or different ethnic groups are needed to confirm these results.

Abbreviations

KD, Kawasaki disease; MCLS, mucocutaneous lymph node syndrome; ITPKC, inositol 1, 4, 5-trisphosphate 3-kinase C; GWAS, genome-wide association studies; WES, whole-exome sequencing; Cis, confidence intervals; MYH14, myosin Heavy Chain 14; RBP3, retinoid-binding protein 3.

Declarations

Authors’ contribution

Xing Zhan MD, Ying Sun BS, Lifen Duan MS, Jianxiao Li MS, and Yanfei Chen MS contribute to supervising the whole project, Xing Zhang MD, Lijuan Meng MS, and Yanfei Chen MS contribute to the design of the study, Caixia Ye BS, Huifeng Han BS, Jianxiao Li MS, Yue Feng MD, and Tiesong Zhang MD contribute to recruit study participants, collect samples, and Xing Zhang MD and Yanfei Chen MS contribute to writing the manuscript.

Acknowledgments

We thank all of our patients and their families for participating in this study. The authors thank Huifeng Han for her contribution to this study. 

Funding

This work was supported by a grant from Kunming "Spring City Plan" High-level talent introduced by the Engineering Young Talents Special Project and a fund project of Yunnan Province Clinical Research Center for Children’s Health and Disease(2022-ETYY-YJ-16).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and tables, and whole-exome sequencing analysis data has been uploaded to NCBI database (No. PRJNA869779).

Ethics approval and consent to participate 

This study was approved by the Ethical Committee, Kunming Children’s Hospital, Yunnan Province under the number 2022/722. 

Consent for publication 

Not applicable. 

Competing interests 

The authors declare that they have no competing interests.

References

  1. Burns JC, Glodé MP. Kawasaki syndrome. The Lancet. 2004;364(9433):533–44.
  2. Kuo HC, Liang CD, Wang CL, Yu HR, Hwang KP, Yang KD. Serum albumin level predicts initial intravenous immunoglobulin treatment failure in Kawasaki disease. Acta Paediatr. 2010;99(10):1578–83.
  3. Sheu JJ, Lin YJ, Chang JS, Wan L, Chen SY, Huang YC, Chan C, Chiu IW, Tsai FJ. Association of COL11A2 polymorphism with susceptibility to Kawasaki disease and development of coronary artery lesions. Int J Immunogenet. 2010;37(6):487–92.
  4. Kwon YC, Kim JJ, Yun SW, Yu JJ, Yoon KL, Lee KY, Kil HR, Kim GB, Han MK, Song MS, et al. BCL2L11 Is Associated With Kawasaki Disease in Intravenous Immunoglobulin Responder Patients. Circ Genom Precis Med. 2018;11(2):e002020.
  5. Marrani E, Burns JC, Cimaz R. How Should We Classify Kawasaki Disease? Front Immunol. 2018;9:2974.
  6. Lin MT, Wang JK, Yeh JI, Sun LC, Chen PL, Wu JF, Chang CC, Lee WL, Shen CT, Wang NK, et al. Clinical Implication of the C Allele of the ITPKC Gene SNP rs28493229 in Kawasaki Disease: Association With Disease Susceptibility and BCG Scar Reactivation. Pediatr Infect Dis J. 2011;30(2):148–52.
  7. Huang YH, Chen KD, Lo MH, Cai XY, Chang LS, Kuo YH, Huang WD, Kuo HC. Decreased DNA methyltransferases expression is associated with coronary artery lesion formation in Kawasaki disease. Int J Med Sci. 2019;16(4):576–82.
  8. Huang YH, Lo MH, Cai XY, Liu SF, Kuo HC. Increase expression of CD177 in Kawasaki disease. Pediatr Rheumatol Online J. 2019;17(1):13.
  9. Kuo HC, Chang WC. Genetic polymorphisms in Kawasaki disease. Acta Pharmacol Sin. 2011;32(10):1193–8.
  10. Onouchi Y. The genetics of Kawasaki disease. Int J Rheum Dis. 2018;21(1):26–30.
  11. Chen MR, Kuo HC, Lee YJ, Chi H, Li SC, Lee HC, Yang KD. Phenotype, Susceptibility, Autoimmunity, and Immunotherapy Between Kawasaki Disease and Coronavirus Disease-19 Associated Multisystem Inflammatory Syndrome in Children. Front Immunol. 2021;12:632890.
  12. Chen SY, Wan L, Huang YC, Sheu JJ, Lan YC, Lai CH, Lin CW, Chang JS, Tsai Y, Liu SP, et al. Interleukin-18 gene 105A/C genetic polymorphism is associated with the susceptibility of Kawasaki disease. J Clin Lab Anal. 2009;23(2):71–6.
  13. Fu LY, Qiu X, Deng QL, Huang P, Pi L, Xu Y, Che D, Zhou H, Lu Z, Tan Y, et al: The IL-1B Gene Polymorphisms rs16944 and rs1143627 Contribute to an Increased Risk of Coronary Artery Lesions in Southern Chinese Children with Kawasaki Disease. J Immunol Res 2019, 2019:4730507.
  14. Kim KY, Bae YS, Ji W, Shin D, Kim HS, Kim DS. ITPKC and SLC11A1 Gene Polymorphisms and Gene-Gene Interactions in Korean Patients with Kawasaki Disease. Yonsei Med J. 2018;59(1):119–27.
  15. Peng Q, Chen C, Zhang Y, He H, Wu Q, Liao J, Li B, Luo C, Hu X, Zheng Z, et al. Single-nucleotide polymorphism rs2290692 in the 3'UTR of ITPKC associated with susceptibility to Kawasaki disease in a Han Chinese population. Pediatr Cardiol. 2012;33(7):1046–53.
  16. Khor CC, Davila S, Breunis WB, Lee YC, Shimizu C, Wright VJ, Yeung RS, Tan DE, Sim KS, Wang JJ, et al. Genome-wide association study identifies FCGR2A as a susceptibility locus for Kawasaki disease. Nat Genet. 2011;43(12):1241–6.
  17. Fujita Y, Nakamura Y, Sakata K, Hara N, Kobayashi M, Nagai M, Yanagawa H, Kawasaki T. Kawasaki disease in families. Pediatrics. 1989;84(4):666–9.
  18. Hata A, Onouchi Y. Susceptibility genes for Kawasaki disease: toward implementation of personalized medicine. J Hum Genet. 2009;54(2):67–73.
  19. Hsu JSJ, So M, Tang CSM, Karim A, Porsch RM, Wong C, Yu M, Yeung F, Xia H, Zhang R, et al. De novo mutations in Caudal Type Homeo Box transcription Factor 2 (CDX2) in patients with persistent cloaca. Hum Mol Genet. 2018;27(2):351–8.
  20. Kim JJ, Hong YM, Yun SW, Lee KY, Yoon KL, Han MK, Kim GB, Kil HR, Song MS, Lee HD, et al. Identification of rare coding variants associated with Kawasaki disease by whole exome sequencing. Genomics Inf. 2021;19(4):e38.
  21. Lo MS. A framework for understanding Kawasaki disease pathogenesis. Clin Immunol. 2020;214:108385.
  22. Kircher M, Witten DM, Jain P, O'Roak BJ, Cooper GM, Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014;46(3):310–5.
  23. Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M. CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 2019;47(D1):D886–94.
  24. Lee YC, Kuo HC, Chang JS, Chang LY, Huang LM, Chen MR, Liang CD, Chi H, Huang FY, Lee ML, et al. Two new susceptibility loci for Kawasaki disease identified through genome-wide association analysis. Nat Genet. 2012;44(5):522–5.
  25. Kim JJ, Yun SW, Yu JJ, Yoon KL, Lee KY, Kil HR, Kim GB, Han MK, Song MS, Lee HD, et al. A genome-wide association analysis identifies NMNAT2 and HCP5 as susceptibility loci for Kawasaki disease. J Hum Genet. 2017;62(12):1023–9.
  26. Wang CL, Wu YT, Liu CA, Kuo HC, Yang KD. Kawasaki disease: infection, immunity and genetics. Pediatr Infect Dis J. 2005;24(11):998–1004.
  27. Uehara R, Belay ED. Epidemiology of Kawasaki disease in Asia, Europe, and the United States. J Epidemiol. 2012;22(2):79–85.
  28. Chen P, Miyake M, Fan Q, Liao J, Yamashiro K, Ikram MK, Chew M, Vithana EN, Khor CC, Aung T, et al. CMPK1 and RBP3 are associated with corneal curvature in Asian populations. Hum Mol Genet. 2014;23(22):6129–36.
  29. Bae Y, Shin D, Nam J, Lee HR, Kim JS, Kim KY, Kim DS, Chung YJ. Variants in the Gene EBF2 Are Associated with Kawasaki Disease in a Korean Population. Yonsei Med J. 2018;59(4):519–23.
  30. Dergun M, Kao A, Hauger SB, Newburger JW, Burns JC. Familial occurrence of Kawasaki syndrome in North America. Arch Pediatr Adolesc Med. 2005;159(9):876–81.
  31. Kwon YC, Kim JJ, Yun SW, Yu JJ, Yoon KL, Lee KY, Kil HR, Kim GB, Han MK, Song MS, et al. Male-specific association of the FCGR2A His167Arg polymorphism with Kawasaki disease. PLoS ONE. 2017;12(9):e0184248.
  32. Arno G, Hull S, Robson AG, Holder GE, Cheetham ME, Webster AR, Plagnol V, Moore AT. Lack of Interphotoreceptor Retinoid Binding Protein Caused by Homozygous Mutation of RBP3 Is Associated With High Myopia and Retinal Dystrophy. Invest Ophthalmol Vis Sci. 2015;56(4):2358–65.
  33. Yokomizo H, Maeda Y, Park K, Clermont AC, Hernandez SL, Fickweiler W, Li Q, Wang CH, Paniagua SM, Simao F, et al. Retinol binding protein 3 is increased in the retina of patients with diabetes resistant to diabetic retinopathy. Sci Transl Med. 2019;11(499):eaau6627.
  34. Wu Q, Blakeley LR, Cornwall MC, Crouch RK, Wiggert BN, Koutalos Y. Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments. Biochemistry. 2007;46(29):8669–79.
  35. Gonzalez-Fernandez F. Interphotoreceptor retinoid-binding protein–an old gene for new eyes. Vis Res. 2003;43(28):3021–36.
  36. Zhu Z, Peng L, Chen G, Jiang W, Shen Z, Du C, Zang R, Su Y, Xie H, Li H, et al. Mutations of MYH14 are associated to anorectal malformations with recto-perineal fistulas in a small subset of Chinese population. Clin Genet. 2017;92(5):503–9.