SLC8A1 Gene Polymorphism Rs13017968 and Hematological Parameters in Kawasaki Disease

Evdoxia Sapountzi^1*, Andreas Giannopoulos¹, Styliani Fidani¹, Maria Trachana², Fragiskos Bersimis³, Spiros Gerou⁴ and Assimina Galli-Tsinopoulou^1
*Correspondence: Evdoxia Sapountzi, 2^nd Department of Paediatrics, School of Medicine, Aristotle University of Thessaloniki, AHEPA University General Hospital, Thessaloniki, Greece, Tel: 00306946465101, Email:

Keywords

Mean platelet volume • Coronary aneurysm • Kawasaki • Single nucleotide polymorphism

Abbreviations

ANOVA: Analysis of Variance; CAA: Coronary Artery Aneurysms; CRP: C-Reactive Protein; ECG: Electrocardiogram; ESR: Erythrocyte Sedimentation Rate; IVIG: Intravenous Immunoglobulin; KD: Kawasaki Disease; LYM: Lymphocytes; MPV: Mean Platelet Volume; MPVLR: Mean Platelet Volume-to-Lymphocyte Ratio; RT-PCR: Real-Time Polymerase Chain Reaction; SD: Standard Deviation; SNP: Single- Nucleotide Polymorphism

Introduction

Kawasaki Disease (KD) is an acute febrile and systemic vasculitis disease mainly affecting children <5 years old [1]. Since the first case of KD, reported in 1967 [1], extensive research has been performed on KD. Nevertheless, the cause of the disease is yet to be deciphered. Coronary artery lesions constitute the most common complications of KD and can significantly increase the risk of coronary heart disease. Coronary Artery Aneurysms (CAA) occur in up to 30% of untreated patients and can lead to ischemic heart disease, myocardial infarction, and even death [2]. The standard treatment for KD is high-dose Intravenous Immunoglobulin (IVIG) plus aspirin within 10 days from symptoms’ appearance, which decreases the incidence of CAA to 5–7% [3-5]. Regardless, around 25% of the patients treated with IVIG become resistant or unresponsive, which increases the risk for CAA development [6].

Both non-genetic and genetic factors have been associated with KD susceptibility and IVIG unresponsiveness. Among the non-genetic factors, certain clinical features and laboratory factors such as a high erythrocyte sedimentation rate (ESR), low hemoglobin and platelet counts, oral mucosa alterations, cervical lymphadenopathy, extremities’ swelling, and polymorphous rash have been linked to IVIG resistance [7]. Moreover, the Mean Platelet Volume-To-Lymphocyte Ratio (MPVLR) was found to be lower in patients with KD [8], whereas a high MPVLR (≥ 2.78) was identified as an independent risk predictor for IVIG resistance in infants with KD under 1 year [9].

Among the genetic factors, polymorphisms in various genes such as ITPKC and CASP3, encoding for inositol-trisphosphate 3-kinase C and caspase 3, respectively [10-15]; cytokine-encoding and cytokine-related genes [6,16-19], Fcγ receptors [20-22]; genes involved in the TGF-γ pathway [12,18,23]; and PLA2G7 [18,24,25], to name a few, have been associated with KD susceptibility and IVIG resistance (for a comprehensive review of polymorphisms affecting IVIG resistance, see [26]).

In this study, we evaluated polymorphism rs13017968 in SLC8A1, encoding for solute carrier family 8, member 1, and various hematological parameters, including MPVLR, as predictors of KD. A previous study by Shimizu C, et al. [27] identified 3 Single-Nucleotide Polymorphisms (SNPs) in SLC8A1 that were associated with KD susceptibility in a Japanese cohort, among which rs13017968 was associated with a higher probability of CAA development and coronary vasodilation. These findings suggest that SLC8A1 rs13017968 may influence both KD susceptibility and the risk of aneurysm formation and dilatation. Hence, we hypothesized that rs13017968 may play a role in the development of KD.

Materials and Methods

Study design

This case-control study was conducted in the Department of Pediatrics of AHEPA University Hospital (Thessaloniki, Greece) and was approved by the corresponding Ethical Review Board (Bioethics Committee, Protocol No. 2.623, 2/27.2.2019). The patients’ parents or legal guardians provided written informed consent prior to study data collection.

Selection criteria

The study included 59 children aged up to 16 years who were diagnosed with KD based on the criteria of the American Heart Association for classic or complete, incomplete, and atypical Kawasaki [4] and treated in our department. Patients with the following characteristics were excluded from the study: history of hepatic disease, history of hematologic disease, malignancy, comorbidity of chronic inflammatory disease, history of chronic itching or eczema, receipt of medication that influence the hematologic parameters. Upon diagnosis, all patients received IVIG infusion at 2 g/kg for 12 hours.

One hundred and seven healthy children aged 6 to 16 years who were registered in the emergency unit were enrolled as control subjects. The selection of this age group was based on the fact that KD rarely occurs in children older than 5 years. Children with a recent history of fever or a medical history of KD at a younger age were excluded.

Outcome measures

Primary outcomes included the frequency of the genetic polymorphism rs13017968 in SLC8A1 and comparison of the MPVLR in patients and control subjects, as well as in patients with and without CAA.

Secondary outcomes included the comparison of the MPVLR before and after the initial IVIG therapy in patients with and without CAA and correlation of the MPVLR with markers of inflammation, age, and sex.

Data collection

For every patient enrolled in the study, the following data were collected: patient demographics (sex, race, birth date, age in months), presence of the genetic polymorphism rs13017968 in SLC8A1. Additionally, the following were recorded before and after IVIG therapy: hematologic parameters (white blood cell count and type, platelet count, hemoglobulin levels, hematocrit, Mean Platelet Volume (MPV), platelet distribution width, and red blood cell distribution width), the levels of acute phase proteins (C Reactive Protein [CRP], ESR), and the MPVLR. All patients were subjected to classic Electrocardiogram (ECG) with Doppler imaging for the assessment of coronary vessels.

The following were recorded for all control subjects: demographics (sex, race, birth date, age in months), presence of the genetic polymorphism rs13017968 in SLC8A1, hematologic parameters (white blood cell count and type, platelet count, hemoglobulin levels, hematocrit, mean platelet volume, platelet distribution width, and red blood cell distribution width), and the MPVLR.

Examinations

After the parents’ or legal guardians’ consent, whole blood was collected from both healthy subjects and patients before IVIG treatment. The levels of urea, creatinine, transaminases, and albumin were determined in the serum using standard methods. CRP and ESR were also determined in all patients.

The number and type of white blood cells, platelet count, hemoglobulin, hematocrit, MPV, platelet distribution width, red blood cell distribution width, and MPVLR were determined. The MPVLR was calculated by dividing the MPV with the absolute number of lymphocytes ([LYM], 103/mm³). For patients, the MPVLR was determined before and after the IVIG therapy.

In both patients and control subjects, 2 mL of heparinized venous blood was collected in EDTA tubes and kept at -80°C until use. DNA isolation from the blood samples was performed according to standard methods according to the manufacturer’s instructions by using commercially available kits.

The identification of rs13017968 in SLC8A1 was performed by real-time polymerase chain reaction (RT-PCR) using specific primers targeting the SNP and TaqMan SNP Genotyping Assay (Thermo Fisher Scientific; Waltham, MA). The RT-PCR was performed using the Real-Time PCR QuantStudio 5 (Thermo Fisher Scientific) and the following PCR cycling conditions: 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds.

Coronary vessels evaluation

Classic 2D ultrasound ECG combined with Doppler imaging was performed in all patients to evaluate the coronary vessels and specifically the left coronary artery, anterior descending artery, and circumflex artery, as well as the right coronary artery. The ultrasound ECG remains the method of choice for early identification of aneurysms in coronary vessels in the first 6 weeks of disease onset [28].

The diagnosis criteria for coronary artery aneurysms related to Kawasaki disease are based on the absolute size of coronary vessels according to age [29].

The z-score was used to evaluate the imaging results of coronary arteries. The z-score is widely used and provides an association of the diameter of the coronary vessels with the total body surface, as well as the Standard Deviation (SD) relative to the mean in z units according to specific normograms. The presence of coronary artery aneurysms or ectasia in combination with a z-score >2.5 is considered a pathological finding. Z-score values <2.5 are considered normal. This classification constitutes a rough patient discrimination system and is recommended when KD is still suspected, aiming to reduce the number of patients at high risk of developing coronary aneurisms. The absolute aneurysm size is an indicator of damages that have already been detected at early stages [4,30,31].

The severity of Kawasaki disease is classified into 5 stages according to the ECG findings [30].

Statistical analysis

Sample size calculation

According to the literature [8], MPVLR is expected to be 3.32 with an SD of 2.66 in patients and 5.32 with an SD of 4.83 in control subjects. To determine the difference between the two groups and considering a statistical power of 80% and significance of 5%, we calculated that a total 120 participants were needed, i.e., 60 per group. The sample size was calculated using OpenEpi Version 3.01.

Statistical methods

Quantitative variables are described by using mean and standard deviation, and categorical variables are presented by using frequency and the corresponding percentage [32]. Kolmogorov-Smirnov and Shapiro-Wilk tests were applied to test the normality of quantitative variables’ distributions [33]. The correlation of qualitative variables was assessed using the chi-square test or Fisher’s exact test when the expected frequency was less than 5 [34-36]. For groups’ comparisons concerning the means of quantitative variables, the parametric Student’s t test for independent samples and paired samples was used [37]. In the case of paired samples (pre vs. post therapy), the effect of the independent variables “IVIG dosage” and “CAA” on the differentiation of the average MPVLR values between the two time points was investigated by applying analysis of variance (ANOVA) with a between subjects factor (mixed ANOVA) [38]. For applying mixed ANOVA, the corresponding assumptions were checked, and no particular violation was found (Box's test, Levene's test, etc.). Spearman correlation coefficient was used for assessing the correlation between quantitative variables that were not distributed normally and was validated by Pearson correlation coefficient [39,40]. All statistical analyses were performed using SPSS, and the level of statistical significance was set at 0.05.

Results

Study population characteristics

The research sample consisted of 60 patients with KD (cases) and 107 healthy participants (control). One case was removed as an outlier during data preprocessing; hence, the analyzed sample was 59 patients. The genetic and phenotypic characteristics of the study population are listed in Table 1. The allele of rs13017968 with the highest frequency was GT and that with the lowest frequency was TT, both in patients with KD and in healthy participants. Sex distribution differed slightly between cases and control, while the mean age of participants in the control group (139.38 months) was much higher than that of patients (37.36 months) (Table 1).

The KD-related features and hematological measurements according to the survey design (pre vs post therapy) are listed in Table 2. Most patients had typical symptoms of the disease (79.7%) and quite a few had non-typical ones (18.6%). Only one patient had incomplete KD symptoms. CAA appeared in 33.9% of the patients and pericardial fluid in 22.0%. The complications of ectasia and mitral insufficiency were identified in 45.8% and 15.3% of the patients, respectively. Most patients received one IVIG dose (78.0%). The mean MPVLR decreased after IVIG treatment (Table 2).

Correlation between Kawasaki disease and rs13017968

No statistically significant relationship was detected between the allele of rs13017968 and the occurrence of KD (χ²=0.162, p=0.922, Chi Square test) (Table 3).

Correlation between CAA occurrence and rs13017968

In patients with KD, no statistically significant relationship was detected between the rs13017968 allele and the occurrence of CAA (p=0.096, Fisher's exact test (Figure 1). Nevertheless, there was a trend for a greater probability of CAA in patients carrying the TT allele than in those carrying the GG allele.

Difference in mean MPVLR values between patients and healthy participants

Kolmogorov-Smirnov and Shapiro-Wilk tests showed that the MPVLR was not normally distributed neither between patients and healthy participants nor between patients before and after IVIG therapy; however, due to the sample sizes (n>30), parametric tests could be applied [41]. For investigating differences in the mean MPVLR between patients with KD and healthy participants, an independent samples t-test was applied. The mean MPVLR was significantly lower in patients with KD than in healthy participants (3.56 ± 2.24 vs. 4.58 ± 1.60, t=3.087, p=0.003). To analyze this result further, a corresponding hypothesis test was conducted for the hematological parameters “LYM” and “MPV”. The mean LYM value was statistically significantly higher in patients than in healthy participants (4059.73 ± 2907.29 vs. 2518.93 ± 771.65, t=-3.994, p<0.05). In contrast, the mean MPV value was statistically significantly lower in patients than in healthy children (9.44 ± 1.03 vs. 10.39 ± 0.92, t=-6.056, p<0.05).

Difference in mean MPVLR values before and after IVIG therapy

A paired t-test was used for investigating differences in the mean MPVLR before and after IVIG treatment in patients with KD. IVIG therapy significantly decreased the mean MPVLR in patients (before: 3.56 ± 2.24 vs. after: 2.40 ± 1.03, t=3.963, p<0.01). In further analysis, no statistically significant difference was detected in hematological measurements “LYM” (t=-1.390, p=0.170) and “MPV” (t=0.502, p=0.618) between the two time points. Mixed ANOVA showed no significant interaction (F(1,57)=2.638, p=0.110) between “MPVLR” and “IVIG” (Figure 2, top) and no significant main effect of the number of IVIG doses (F(1,57)=0.043, p=0.837). There was also no significant interaction (F(1,57)=1.090 p=0.301) between “MPVLR” and “CAA” (Figure 2, bottom), and no significant main effect of MPVLR in the CAA group (F(1,57)=0.337, p=0.564). The assumptions for applying mixed ANOVA were satisfied (e.g., Box's Test p>0.05, Levene's Test p>0.05).

Correlation between CAA occurrence and sex in patients with KD

A statistically significant relationship was detected between CAA occurrence and sex (χ²=5.597, p=0.023). In particular, the probability of CAA occurrence was greater among boys than among girls with KD (Figure 3).

Correlation among Age, Pre MPVLR, and Post MPVLR for KD patients

The variables Age, Pre MPVLR, and Post MPVLR did not follow a normal distribution, and no clear linear pattern was visible in a scatter diagram (Figure 4). Therefore, the non-parametric Spearman’s correlation coefficient was used for investigating the potential correlation among them, which was verified by the corresponding parametric Pearson’s correlation coefficient. Age was positively and moderately correlated with the Pre MPVLR value (Spearman's rho=0.543, p<0.05; Table 4) and with the Post MPVLR value (Spearman's rho=0.534, p<0.05; Table 4). In addition, Pre MPVLR and Post MPVLR were also positively and weakly to moderately correlated (Spearman's rho=0.319, p<0.05; Table 4).

Discussion

In the present study, we investigated the relationship between a SNP in SLC8A1, rs13017968, and KD, as well as the impact of hematologic factors in KD expression.

According to a previous study, rs13017968 was identified as one of three SNPs affecting KD susceptibility in a Japanese cohort [27]. The three validated SLC8A1 SNPs (rs10490051, rs13017968, and rs12989852), found in the same linkage disequilibrium block as a cluster of associated SNPs identified by GWAS in a European cohort, are located 80 kb downstream from the splice donor site of the first exon that is shared by all 15 transcript variants of SLC8A1. Patients who were homozygous for the risk allele had a higher risk of developing aneurysms/dilation [27].

The same group [42] utilized expression quantitative trait loci analysis and showed that patients homozygous for rs13017968 or any of the other two risk alleles in SLC8A1 had elevated mRNA and plasma protein levels of urotensin 2, a molecule involved in vasoconstriction (via vascular smooth muscle cells) [43]. Based on the above findings, we postulated a role for rs13017968 in KD. However, when comparing a cohort of children with KD and healthy children, we did not confirm a significant difference in the frequency of rs13017968. Moreover, we could not confirm a significant difference in the frequency of rs13017968 between patients with and those without CAA despite a trend for a higher probability for developing CAA among patients carrying the TT than the GG allele. Possible population differences might explain the discrepancy between our and previous findings.

The MPVLR was found to be significantly lower in patients with KD than in healthy participants, whereas there was no significant interaction between MPVLR and CAA. Previous finding regarding the MPVLR in KD are contradictory, as both lower [8] and higher values have been reported in patients. The lower ratio in our study is justified by the significantly lower MPV values in patients than in controls and the respective significantly higher LYM values.

In agreement, two previous studies by Yu-wei HU, et al. [44] and Liu R, et al. [45] also reported significantly lower MPV in patients with KD than in control subjects. In contrast, Kim SH, et al. [46] showed no differences in the MPV between patients and controls at the time of KD diagnosis. In our study, we did not found an interaction between MPVLR and CAA development, which contradicts previous findings indicating lower MPVLR in patients with CAA [8]. However, consistently with previous studies [46], IVIG treatment decreased the mean MPVLR in our patient cohort. The children with KD in our study were significantly younger than the included healthy children, and we found that age significantly correlated with the MPVLR both before and after IVIG therapy. Therefore, potential differences might be explained by different population characteristics.

CAA is an important and the most potentially debilitating complication of KD. In our study, 33.9% of the patients had developed CAA. A significant association was identified between sex and the occurrence of CAA Specifically; boys were at higher risk of developing CAA, in agreement with the findings of previous studies [47-50].

An important limitation of the present study is the relatively small sample size of patients, which did not allow the use of parametric hypothesis tests to investigate all of the research questions. Moreover, the patients were enrolled from a single center. Nonetheless, the rarity of the disease justifies the small sample, particularly in a small country like Greece. Future similar investigations on a larger scale may be necessary to address the involvement of rs13017968 in KD and CAA formation, as well as the significance of the MPVLR in KD and/or CAA risk prediction. The identification of such genetic or hematological predictors is crucial in determining appropriate treatment plans for patients.

Conclusion

Overall, our results provide insight on KD characteristics and association with rs13017968 and hematological parameters in a small sample in Greece. Further, larger sample studies are needed to verify our results.

Competing Interests

The authors have no competing interests to declare that are relevant to the content of this article.

Funding

No funding was received to assist with the preparation of this manuscript.

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