Identification of LEF1 as a Susceptibility Locus for Kawasaki Disease in Patients Younger than 6 Months of Age

Article information

Genomics Inform. 2018;16(2):36-41

Publication date (electronic) : 2018 June 30

doi : https://doi.org/10.5808/GI.2018.16.2.36

Hea-Ji Kim¹, Sin Weon Yun², Jeong Jin Yu³, Kyung Lim Yoon⁴, Kyung-Yil Lee⁵, Hong-Ryang Kil⁶, Gi Beom Kim⁷, Myung-Ki Han⁸, Min Seob Song⁹, Hyoung Doo Lee¹⁰, Kee Soo Ha¹¹, Sejung Sohn¹², Ryota Ebata¹³, Hiromichi Hamada¹⁴, Hiroyuki Suzuki¹⁵, Yoichiro Kamatani¹⁶, Michiaki Kubo¹⁷, Kaoru Ito¹⁸, Yoshihiro Onouchi^18,§, Young Mi Hong^12,§, Gi Young Jang^11,§, Jong-Keuk Lee^1,§,, The Korean Kawasaki Disease Genetics Consortium

¹Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea

²Department of Pediatrics, Chung-Ang University Hospital, Seoul 06973, Korea

³Department of Pediatrics, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Korea

⁴Department of Pediatrics, Kyung Hee University Hospital at Gangdong, Seoul 05278, Korea

⁵Department of Pediatrics, Daejeon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Daejeon 34943, Korea

⁶Department of Pediatrics, Chungnam National University Hospital, Daejeon 35015, Korea

⁷Department of Pediatrics, Seoul National University Children’s Hospital, Seoul 03080, Korea

⁸Department of Pediatrics, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung 25440, Korea

⁹Department of Pediatrics, Inje University Busan Paik Hospital, Busan 47392, Korea

¹⁰Department of Pediatrics, Pusan National University Hospital, Busan 49241, Korea

¹¹Department of Pediatrics, Korea University Ansan Hospital, Ansan 15355, Korea

¹²Department of Pediatrics, Ewha Womans University Hospital, Seoul 07985, Korea

¹³Department of Pediatrics, Chiba-University Graduate School of Medicine, Chiba 260-8670, Japan

¹⁴Department of Pediatrics, Tokyo Women’s Medical University Yachivo Medical Center, Yachivo 276-8524, Japan

¹⁵Department of Pediatrics, Wakayama Medical University, Wakayama 641-8509, Japan

¹⁶Laboratory for Statistical Analysis, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan

¹⁷Laboratory for Genotyping Development, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan

¹⁸Laboratory for Cardiovascular Diseases, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan

^*Corresponding author: Tel: +82-2-3010-4142, Fax: +82-2-488-3312, E-mail: cookie_jklee@hotmail.com

§These authors contributed equally to this work.

Received 2018 March 23; Revised 2018 April 11; Accepted 2018 April 11.

Abstract

Introduction

Kawasaki disease (KD) is an acute, self-limited vasculitis that predominantly occurs in children between the ages of 6 months and 5 years old. Approximately half of all KD patients are between 6 months to 2 years of age, which is the peak incidence age of KD [1, 2]. The etiology of KD is not known, and it has no specific diagnostic test. As such, KD diagnosis is based solely on six clinical symptoms: prolonged fever, bilateral conjunctival injection, erythema of the oral mucosa, lips, and tongue, polymorphous rash, erythema of the palms and soles, and cervical lymphadenopathy [3]. Complete KD is diagnosed when patients have at least five of the above six clinical symptoms, and incomplete KD is diagnosed when patients have less than four of the six clinical symptoms. The standard treatment of KD is a high-dose intravenous immunoglobulin (IVIG), which is derived from pooled plasma of healthy donors, reducing the duration of fever and the incidence of coronary artery abnormalities [4, 5].

KD is considered an abnormal immunological reaction to an infection or unknown immunological triggers in genetically susceptible individuals [6, 7]. B cell–related genes including BLK, CD40, and FCGR2A were also identified as KD susceptibility genes by genome-wide association studies (GWAS) [8-10]. In particular, a reduced level of BLK expression in blood B cells may be a crucial reason for dysfunction of B cells and a pathogenesis of KD [10, 11]. Our previous study identified that a risk allele in FCGR2A was only susceptible for KD patients younger than 1 year of age, whereas the KD susceptible allele in BLK affected all ages of KD patients, except those older than 5 years of age. This result revealed a possibility that there are other genetic variants affecting specific age subgroups of KD patients. In this study, to further identify age-specific susceptibility genes of KD in KD patients younger than 6 months or older than 5 years of age, we performed an age-stratified GWAS and a replication study and identified lymphoid enhancer binding factor 1 (LEF1) as a KD susceptibility gene specifically affecting KD patients younger than 6 months of age.

Methods

Results

Identification of LEF1 as a KD susceptibility gene specifically associated with KD in patients younger than 6 months of age.

To identify the genetic variants affecting two extreme age subgroups of KD patients (younger than 6 months and older than 5 years of age), we performed an age-stratified GWAS using 19 KD cases younger than 6 months or 45 KD cases older than 5 years of age, respectively, compared to 1,000 controls. A total of 12 SNPs for patients younger than 6 months and 14 SNPs for those older than 5 years of age were chosen for the replication study on the basis of our arbitrary threshold (p ＜ 1×10^-4 and genes related to immune functions) for each subgroup. Among 26 candidate SNPs tested in the replication study (Supplementary Tables 1 and 2), only a nonsynonymous SNP (rs4365796: c.1106C＞T, p.Thr369Met) in the LEF1 gene was validated in Korean KD patients younger than 6 months of age (odds ratio [OR], 5.92; p = 0.000268 in GWAS and OR, 2.60; p = 0.00126 in the replication study) (Table 1). The combined analysis of the Korean GWAS and replication for LEF1 SNP (rs4365796) showed very significant association with KD in patients younger than 6 months of age (118 cases vs. 4,553 controls; OR, 3.07; p_combined = 1.10 × 10^-5), whereas no association of the same SNP was observed in any other age group of KD patients (Fig. 1, Supplementary Table 3). To further validate our findings in another population, we also performed a replication study in the Japanese cohort comprised of 1,306 KD cases and 6,893 controls. The SNP rs4365796 in the LEF1 gene showed the same direction of risk effect in Japanese KD patients younger than 6 months of age, although the effect was statistically not significant (OR, 1.42; p = 0.397) (Table 1). In a meta-analysis of Korean and Japanese data, a significant association was observed in KD patients younger than 6 months of age (OR, 2.50; p = 5.01 × 10^-5), whereas no association was detected in KD patients older than 6 months of age (OR, 1.10; p = 0.342) (Table 1). This result indicates that the LEF1 gene is a novel susceptibility gene specifically affecting KD patients younger than 6 months of age.

Table 1.

Age-stratified association results for LEF1 (rs4365796; risk allele: T) in Korean and Japanese populations

Fig. 1.

Odds ratios (ORs) and confidence intervals (CIs) of the LEF1 (rs4365796) association with Kawasaki disease (KD) according to age of Korean KD patients (total number of KD patients, 1,658). Each horizontal bar is a 95% CI. A total of 4,553 controls were used in the genetic association analysis for each age subgroup of KD patients. Significant p-values (p < 0.05) are shown in bold.

To determine the effect of the LEF1 risk allele on clinical features of KD patients, we examined the clinical data classified by the LEF1 genotypes (rs4365796, risk allele: T) in 1,656 Korean KD patients (number of genotype: CC = 1,559, CT/TT = 96/1). When we investigated the effect of genotype of the LEF1 risk allele in two KD subgroups (younger than 6 months and older than 6 months of age), the risk allele did not show any significant effect on any clinical variables in KD patients with one exception; the risk genotypes (either CT or TT) of the LEF1 gene (rs4365796) had slightly increased CRP levels (median, 8.48 mg/L; p = 0.032) in KD patients older than 6 months of age compared to the non-risk genotype (CC) of the LEF1 gene (median, 6.50 mg/L) (Supplementary Table 4).

Discussion

The incidence of KD in patients younger than 6 months and older than 5 years of age was comparatively lower than other age groups. The peak incidence of KD was in the age group between 6 months and 2 years of age. KD has no diagnostic test and the diagnosis depends on clinical symptoms. In particular, KD patients younger than 6 months of age can be difficult to diagnose because they show fewer clinical symptoms, called incomplete KD, which leads to delayed diagnosis or misdiagnosis in infants. Consequently, cardiac complications are more common in KD patients younger than 6 months than in older children [16-18]. Therefore, early and accurate diagnosis of infantile KD is important to reduce the risk of cardiac complications. In this study, we analyzed GWAS data of patients younger than 6 months and older than 5 years of age to identify new risk loci for KD susceptibility in these age groups. We identified a new KD susceptibility locus in the LEF1 gene (rs4365796: c.1106C＞T, p.Thr369Met) on chromosome 4. The associated amino acid-altering SNP (rs4365796) in the LEF1 gene had an unusually strong effect size, a 3.07-fold increased risk for incidence of KD, in Korean KD patients younger than 6 months of age. This result indicates that the LEF1 gene plays a crucial role in the pathogenesis of KD in very young children and this amino acid-altering variant can be used as a candidate marker to identify high risk KD patients in infant patients in a clinical setting.

The LEF1 gene encodes a transcription factor that is expressed in developing B and T cells and at multiple sites of organogenesis during embryonic development [19-21]. LEF1 is a central mediator of the Wnt signaling pathway through recruiting β-catenin and plays crucial roles during development, including normal hematopoiesis [22, 23]. Abnormal protein expression of LEF1 has been detected in chronic lymphocytic leukemia (CLL) cells and monoclonal B-cell lymphocytosis, indicating that LEF1 plays an early role in B-cell development and CLL leukemogenesis [24]. Transplantation of LEF1-transduced bone marrow also developed acute myeloid leukemia and B-precursor acute lymphoblastic leukemia in mouse models [25]. Additionally, LEF1 contributes to the survival and proliferation of pro-B cells during early B cell development [26]. GWAS also identified the LEF1 gene as a susceptibility locus for systemic lupus erythematosus and CLL [27-29]. These previous results suggest that the dysfunction of the LEF1 gene is involved in early B lymphocyte development, which is involved in the pathogenesis of KD. In our study, we found that a nonsynonymous SNP (rs4365796: c.1106C＞T, p.Thr369Met) was significantly associated with KD in patients younger than 6 months of age. This SNP was predicted as probably damaging and deleterious by PolyPhen and SIFT, respectively, suggesting that this amino acidaltering variant can change the biological functions of LEF1 protein, probably during the early development of B cells.

Although we found that a nonsynonymous SNP (rs4365796: c.1106C＞T, p.Thr369Met) in the LEF1 gene is significantly associated with KD in Korean patients younger than 6 months of age, the biological role of the LEF1 variant is still unknown. Therefore, we should investigate how the amino acid-altering LEF1 variant specifically affects the immune response in infants and subsequently the potential mechanism of LEF1-mediated pathogenesis in KD. Additionally, the significantly associated SNP (rs4365796) in the LEF1 gene in Korean KD patients was not replicated in the Japanese samples, although the same direction of risk effect was detected (Table 1). As shown in Table 1, the risk allele of the LEF1 gene is a rare variant with lower allele frequency in the Japanese population (risk allele frequency = 0.023 in Korean vs. 0.018 in Japanese control samples). Furthermore, the portions of KD patients younger than 6 months of age was higher in Japan (9.19% in Japan vs. 7.13% in Korea) (Table 1), suggesting that Korean KD patients younger than 6 months of age are a genetically enriched and more homogeneous case population. A previous study also reported that the Japanese population has a higher incidence of KD in patients younger than 6 months of age compared to the Korean population (11.2% in Japanese KD patients vs. 7.7% in Korean KD patients) [30]. Therefore, we assume that no replication of the LEF1 gene in the Japanese samples may be due to the lower frequency of the risk allele in the LEF1 gene and/or higher KD incidence in those younger than 6 months of age compared to the Korean population. Conversely, another limitation of our study is the low statistical power due to the small sample size resulting from the selection of a rare variant with an allele frequency less than 2.5% and of age subgroups of KD patients, particularly younger than 6 months of age (less than 10% of total KD cases). Therefore, to support our findings, further replication studies are necessary in more independent sample sets with larger sample size.

In conclusion, we identified that a nonsynonymous SNP (rs4365796: c.1106C＞T, p.Thr369Met) in the LEF1 gene is significantly associated with KD in children younger than 6 months of age. This amino acid-altering variant in the LEF1 gene will be useful to identify high risk KD patients younger than 6 months of age because this SNP had a strong effect size (OR, 3.07). This result will provide new insight into the pathogenesis of the KD in infants.

Supplementary Materials

Article information Continued