Application of rab31 / rs9965664 in evaluating immunoglobulin resistance of patients with kawasaki disease

Abstract

The application discloses application of Rab31 / rs9965664 in evaluation of immunoglobulin drug resistance of Kawasaki disease patients; the application finds that KD patients carrying Rab31 / rs9965664 AA / GA genotypes are more likely to have IVIG drug resistance, and the risk of IVIG treatment resistance is significantly increased, so the IVIG drug resistance can be evaluated by detecting the Rab31 / rs9965664 genotype; it is also found that the Rab31 protein expression level in mononuclear cells of KD patients carrying the rs9965664 A allele is generally low, and the Rab31 protein expression level in mononuclear cells of patients in the IVIG treatment resistance group is significantly lower than that in the IVIG sensitive group, so the IVIG drug resistance risk can also be evaluated by detecting the Rab31 expression amount. It is favorable for follow-up treatment of the Kawasaki disease patients and has high application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of Rab31 / rs9965664 in evaluation of immunoglobulin resistance of Kawasaki disease patients. BACKGROUND

[0002] Kawasaki disease (KD) is an acute febrile systemic vasculitis that mainly affects infants and young children. High-dose intravenous immunoglobulin (IVIG) is the first-line treatment for KD patients to reduce inflammation and the risk of coronary artery aneurysm (CAA) formation. Unfortunately, 10-20% of patients do not respond to this treatment, and such patients are defined as IVIG-resistant. In addition, many studies have found that Rab31 regulates the innate immune response in a variety of human diseases. However, there is no report on whether single nucleotide polymorphism (SNP) of Rab31 gene is related to IVIG resistance of KD patients.

[0003] Kawasaki disease (KD) is an acute febrile systemic vasculitis that mainly affects infants and young children. High-dose intravenous immunoglobulin (IVIG) is the first-line treatment for KD patients to reduce inflammation and the risk of coronary artery aneurysm (CAA) formation. Unfortunately, 10-20% of patients do not respond to this treatment, and such patients are defined as IVIG-resistant. In addition, many studies have found that Rab31 regulates the innate immune response in a variety of human diseases. However, there is no report on whether single nucleotide polymorphism (SNP) of Rab31 gene is related to IVIG resistance of KD patients.

[0004] Intravenous immunoglobulin (IVIG) is a product purified from the plasma of thousands of donors and is widely used in a variety of autoimmune and inflammatory diseases, such as immune thrombocytopenia (ITP) and KD. At present, the high-dose IVIG treatment regimen is still the first-line treatment for KD patients, because IVIG treatment can reduce the incidence of persistent inflammation and the risk of sudden death caused by coronary artery aneurysm (CAA) formation. Unfortunately, 10-20% of patients do not respond to this treatment and eventually develop IVIG resistance. IVIG-resistant patients have an increased risk of developing life-threatening complications such as Kawasaki disease shock syndrome (KDSS) or KD macrophage activation syndrome (KD-mas) compared to IVIG-sensitive patients. Therefore, understanding the mechanism of action of IVIG is crucial for the clinical treatment of KD.

[0005] Human Rabs (Ras-related proteins in brain) belong to the Ras family of small GTPases, with more than 60 family members. The function of Rabs depends on their molecular switching between GTP-on and GDP-off states, and the regulation of specific membrane transport in cellular endocytosis and exocytosis. Rab31 is also an important subgroup of the Rab superfamily, mainly located in the Golgi complex and organelle membranes derived from the Golgi apparatus. In addition to membrane transport function, Rab31 also regulates the heterogeneity of exosome biogenesis by recruiting TBC1D2B. In addition, some documents also report that Rab31 is also essential for innate immunity. Yeo et al. found that Rab31 recruits signal adaptor APPL2 in the early phagocytic PI(4,5)P2 to PI(3,4,5)P3 conversion process, and then regulates macrophage fcγ receptor (FcγR) mediated phagocytosis through the PI3K signaling pathway.

[0006] Macrophage phagocytosis is essential for inflammatory and autoimmune responses, and can remove pathogens and tissue debris. More interestingly, the mechanism of action of IVIG in immune thrombocytopenia and hemolytic anemia is to prevent autoantibody-mediated phagocytosis of blood cells by competing with splenic and hepatic macrophage FcγRs. It is suggested that Rab31 regulation of macrophage-related phagocytosis may be the key to IVIG action, and the role of Rab31 in KD or IVIG has so far remained blank. SUMMARY

[0007] The purpose of the present application is to provide the application of Rab31 / rs9965664 in evaluating the immunoglobulin resistance of Kawasaki disease patients.

[0008] The technical solution adopted by the present application is:

[0009] In a first aspect of the present application, the application of a substance for detecting the expression amount of Rab31 in the preparation of a Kawasaki disease immunoglobulin resistance evaluation product is provided.

[0010] In some embodiments of the present application, the expression amount of Rab31 is decreased in the immunoglobulin resistance of Kawasaki disease.

[0011] In some embodiments of the present application, the product includes a kit, a diagnostic reagent, a detection reagent, a test paper, a gene chip or a protein chip.

[0012] In some embodiments of the present application, the substance includes a substance for detecting the expression amount of Rab31 by immunoblotting, immunohistochemical method and immunoprecipitation method, enzyme-linked immunosorbent assay, colloidal gold, mass spectrometry and the like.

[0013] In some embodiments of the present application, the substance includes reagents, instruments, software and / or modules for detecting Rab31 expression level.

[0014] In a second aspect of the present application, there is provided use of a substance for detecting rs9965664 polymorphism or genotype in the preparation of a product for evaluating immunoglobulin resistance of Kawasaki disease.

[0015] In some embodiments of the present application, the presence of AA / GA genotype of rs9965664 indicates an increased risk of immunoglobulin resistance of Kawasaki disease, decreased sensitivity and easy occurrence of treatment resistance.

[0016] In some embodiments of the present application, male patients and / or patients less than 60 months old with AA / GA genotype of rs9965664 are more likely to have an increased risk of immunoglobulin resistance of Kawasaki disease, decreased sensitivity and easy occurrence of treatment resistance.

[0017] In some embodiments of the present application, the product includes a kit, a diagnostic reagent, a detection reagent, a test paper, a gene chip or a protein chip.

[0018] In some embodiments of the present application, the substance for detecting rs9965664 includes a substance for detecting rs9965664 polymorphism or genotype by TaqMan probe method, SNaPshot method, HRM method, Mass Array method or Illumina BeadXpress method.

[0019] In some embodiments of the present application, the substance includes reagents, instruments, software and / or modules for detecting rs9965664 polymorphism or genotype.

[0020] In some embodiments of the present application, the substance includes primers and probes.

[0021] In a third aspect of the present application, there is provided a method for detecting immunoglobulin resistance of Kawasaki disease for non-diagnostic purposes, by Rab31 expression level and / or rs9965664 polymorphism or genotype of a Kawasaki disease patient.

[0022] In some embodiments of the present application, Rab31 expression level is decreased in immunoglobulin resistance of Kawasaki disease, and when its expression level is lower than that of a normal person, it indicates an increased risk of immunoglobulin resistance of Kawasaki disease.

[0023] In some embodiments of the present application, the presence of AA / GA genotype of rs9965664 indicates an increased risk of immunoglobulin resistance of Kawasaki disease, decreased sensitivity and easy occurrence of treatment resistance.

[0024] In a fourth aspect, the present application provides a method for screening a treatment for Kawasaki disease immunoglobulin drug resistance, by detecting the Rab31 expression and / or rs9965664 polymorphism or genotype of a Kawasaki disease patient before and after administration.

[0025] The present application has the following beneficial effects:

[0026] The present application finds that KD patients carrying Rab31 / rs9965664 AA / GA genotype are more likely to develop IVIG resistance than KD patients carrying GG genotype, and the risk of IVIG treatment resistance is significantly increased, and the difference is statistically significant, so the IVIG resistance can be evaluated by detecting the Rab31 / rs9965664 genotype. In addition, the present application also finds that the expression level of Rab31 protein in mononuclear cells of KD patients carrying rs9965664 A allele is generally lower, and the expression level of Rab31 protein in mononuclear cells of patients in the IVIG treatment resistance group is significantly lower than that in the IVIG sensitive group, which is consistent with the foregoing results, so the IVIG resistance risk can also be evaluated by detecting the expression level of Rab31; It is beneficial to the follow-up treatment of Kawasaki disease patients and has high application value. Moreover, mechanism research shows that Rab31 may regulate the response of Kawasaki disease patients to IVIG treatment through NLRP3 and p38 signaling pathways. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Rab31 regulates the response of Kawasaki IVIG treatment through NLRP3 / P38 signaling pathway.

[0028] Figure 1 (A) Negative correlation between Rab31 expression and phosphorylated P38 expression in PBMC of Kawasaki IVIG sensitive group patients; Figure 1 (B) Positive correlation between Rab31 expression and phosphorylated P38 expression in PBMC of Kawasaki IVIG resistance group patients; Figure 1 (C) Protein expression diagram of NLRP3, Rab31, phosphorylated P38 and other proteins in IVIG resistance and IVIG sensitive groups; Figure 1 (D) Expression level statistical diagram of NLRP3, Rab31, phosphorylated P38 and other proteins in IVIG resistance and IVIG sensitive groups; Figure 1 (E) Rab31 expression diagram in PBMC of Kawasaki disease patients carrying different genotypes (Mutation is AA genotype, Wildtype is GG genotype); Figure 1 (F) Rab31 expression level statistical diagram in PBMC of Kawasaki disease patients carrying different genotypes. DETAILED DESCRIPTION

[0029] The concept and technical effects of the present application will be described clearly and completely in combination with the embodiments below, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all embodiments, and other embodiments obtained by those skilled in the art on the basis of the embodiments of the present application without creative labor are within the scope of protection of the present application.

[0030] Materials and methods

[0031] 1. Sample information

[0032] From January 2014 to December 2021, a total of 1024 KD patients were recruited from Guangzhou Women and Children's Medical Center. The accuracy of the diagnosis and treatment plan of all participating KD patients was confirmed by reviewing the hospital database and echocardiogram database information.

[0033] 2. Clinical diagnosis of Kawasaki disease

[0034] All KD patients were diagnosed by pediatricians according to the American Heart Association criteria.

[0035] 3. IVIG treatment response

[0036] The criteria for IVIG treatment response are: within 11 days of the initial fever, after the first use of IVIG (2g / kg) treatment, patients still have persistent or recurrent fever (temperature >= 38.0℃, axillary or oral body temperature) for more than 36 hours, and the total time is less than 7 days. Patients are determined to be IVIG resistant.

[0037] 4. Genotyping and DNA extraction

[0038] Peripheral blood of KD patients was collected. Genomic DNA was extracted using the TIANamp Blood DNA Kit (DP318, TIANGEN Biotech, Beijing) and operated according to the manufacturer's instructions. The rs9965664 specific fluorescent allele probe was purchased from ABI Company (Thermo Fisher Scientific, United States). PCR was performed on a 384-well plate using the ABI-Q6 sequence detection system (Thermo Fisher Scientific).

[0039] SNP genotyping was performed using TaqMan SNP Genotyping (Cat No: 4351379, Thermofisher). Laboratory technicians were blind to sample information, including specific information related to duplicate samples. 10% of the duplicate genotyping results of two randomly selected sample groups were selected. The coincidence rate was 100%.

[0040] 5. Immunoblotting

[0041] PBMC total protein was extracted from cells using cell lysis solution containing PMSF (Beyotime). The samples were separated by 12.5% sds-polyacrylamide gel electrophoresis and transferred to PVDF membrane. After blocking the membrane in PBS containing 5% bovine serum albumin, it was incubated with Rab31 primary antibody (1:1000; 16182-1-AP, Proteintech), NLRP3 antibody (1:1000; 15101, Cell Signaling Technology), p-P38 antibody (1:1000; ARG51850, Arigo), or β-actin antibody (1:500; 380624, Zen BioScience) at 4°C overnight, and then with peroxidase-conjugated secondary antibody (anti-rabbit IgG, hrp-conjugated, 1:500; 7074S, CST) or anti-mouse IgG, hrp-conjugated antibody, 1:2000; 7076S, CST) at room temperature for 1 hour. The signal was then detected by x-ray film. All images were analyzed using ImageJ.

[0042] 6. Statistical methods

[0043] SAS software (version 9.4; SAS Institute, Cary, North Carolina) was used. Pearson’s chi-squared test was used to assess differences in demographic variable distribution and genotype frequencies between IVIG-sensitive and IVIG-resistant cases. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression analysis to measure the association between the Rab31 / rs9965664 polymorphism and the risk of IVIG resistance in KD patients. Relative protein expression fold change statistics were plotted using GraphPad Prism 8. A p-value <0.05 was considered statistically significant.

[0044] Results

[0045] 1. Clinical characteristics of KD patients included in the analysis

[0046] A total of 1024 KD patients were included in this study (Table 1).

[0047] Table 1. Distribution of characteristics of KD patients in IVIG-resistant and -sensitive groups

[0048]

[0049] Table 1 shows the characteristic distribution of 817 IVIG-sensitive KD patients and 207 IVIG-resistant KD patients. The proportion of IVIG-resistant patients was 20.2%, consistent with previous studies. The mean age of the IVIG-sensitive group was 25.68 ± 20.65 months (range 1–131 months), and the mean age of the IVIG-resistant group was 27.69 ± 22.68 months (range 2–132 months). Among the IVIG-sensitive KD patients, 62.55% were male, and among the IVIG-resistant KD patients, the proportion of males was 67.63%. The proportions of females were 37.45% and 32.37%, respectively. There were no significant differences in age (p = 0.189) and sex (p = 0.172) between the IVIG-sensitive and resistant groups.

[0050] 2. Correlation analysis between Rab31 / rs9965664 polymorphism and IVIG resistance

[0051] The genotype frequency distribution of Rab31 / rs9965664 polymorphism in the KD IVIG treatment resistance and sensitivity groups is shown in Table 2. To investigate the association between Rab31 / rs9965664 polymorphism and the risk of IVIG treatment resistance, a χ² test was performed. 2 Analysis revealed that the Rab31 / rs9965664 polymorphism was significantly associated with an increased risk of IVIG resistance (GA vs GG: adjusted OR = 1.45, 95% CI = 1.05–2.00, P = 0.025; AA vs GG: adjusted OR = 2.46, 95% CI = 1.41–4.30, P = 0.002; AA / GA vs. GG: adjusted OR = 1.57, 95% CI = 1.16–2.14, P = 0.004; AA vs. GA / GG: adjusted OR = 2.09, 95% CI = 1.22–3.57, P = 0.007). These results suggest that patients carrying the AA / GA genotype have a significantly higher risk of developing IVIG resistance than those carrying the GG genotype, indicating that this SNP has a resistant effect on IVIG treatment.

[0052] Table 2 Genotype frequency distribution of Rab31 / rs9965664 polymorphism in KD IVIG treatment resistant and sensitive groups

[0053]

[0054] 3. Stratified analysis

[0055] Further analysis was performed to investigate the association of Rab31 / rs9965664 polymorphism with the risk effect of IVIG treatment response in specific subgroups divided by age and gender (Table 3). Male patients carrying rs4594236 AA / GA genotype were more susceptible to IVIG treatment resistance than KD patients carrying rs9965664 / GG genotype (adjusted OR = 1.86, 95% CI = 1.27-2.71, p = 0.001), especially in patients less than 60 months of age (adjusted OR = 1.56, 95% CI = 1.13-2.15, p = 0.006).

[0056] Table 3

[0057]

[0058] 4、Rab31 in IVIG-resistant KD patients monocytes expression decreased

[0059] To investigate the role of Rab31 in KD patients' response to IVIG treatment, Western blotting was performed on 10 KD monocytes (5 IVIG-sensitive and 5 IVIG-resistant). The results showed that Rab31 protein expression in IVIG-resistant group was significantly lower than that in IVIG-sensitive group Figure 1 ).

[0060] The Rab31 Rs9965664 locus is located in the intronic region of chromosome 18, and it is well known that introns can affect the expression of many genes in various ways. To verify whether rs9965664 affects Rab31 protein expression, the Rab31 protein expression level in monocytes of patients with different genotypes of rs9965664 was measured, and the results showed that Rab31 protein expression level in monocytes of patients carrying rs9965664 A allele was lower, which was consistent with the correlation of clinical IVIG treatment results Figure 1 ). Applicants also explored the possible downstream pathway of Rab31, and found that NLRP3 was significantly increased in IVIG-resistant group. There was no difference in the level of phosphorylated p38 between IVIG-sensitive and IVIG-resistant groups, while the level of Rab31 protein was negatively correlated with the level of p-P38 in IVIG-resistant group and positively correlated in IVIG-sensitive group. In summary, Rab31 protein expression level in KD patients carrying rs9965664 A allele was lower, and Rab31 might inhibit the response to IVIG treatment through NLRP3 and p38 signaling pathways.

[0061] The above detailed description of the application has been made with reference to specific embodiments thereof, but the application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the application. Furthermore, the embodiments of the application and the features in the embodiments can be combined with each other in the case of no conflict.

Claims

1. Use of a substance for detecting the expression level of Rab31 protein in the preparation of a product for evaluating immunoglobulin resistance of Kawasaki disease.

2. Use according to claim 1, characterized in that, The expression level of Rab31 protein is decreased in the immunoglobulin resistance of Kawasaki disease.

3. Use according to claim 1, characterized in that, The product comprises a kit, test paper or protein chip.

4. Use of a substance detecting the rs9965664 polymorphism or genotype for the manufacture of a product for the evaluation of immunoglobulin resistance in Kawasaki disease, characterized in that, The presence of AA / GA genotype of rs9965664 indicates an increased risk of immunoglobulin resistance of Kawasaki disease.

5. Use according to claim 4, characterized in that, The product comprises a kit, test paper or gene chip.

6. Use according to claim 4, characterized in that, The substance for detecting the polymorphism or genotype of rs9965664 comprises a substance for detecting the polymorphism or genotype of rs9965664 by TaqMan probe method, SNaPshot method, HRM method, Mass Array method or Illumina BeadXpress method.

7. Use according to claim 6, characterized in that, The substance comprises a primer and a probe.

Images