Identification of HLA-DRB1*04:10 allele as risk allele for Japanese moyamoya disease and its association with autoimmune thyroid disease: A case-control study

Identification of HLA-DRB1*04:10 allele as risk allele for Japanese moyamoya disease and its... Tokunaga K, Fujimura M, Sakata H, et al. (2019) Identification of HLA-DRB1 04:10 allele as risk allele for Japanese moyamoya disease and its association with autoimmune thyroid disease: A Background and purpose case-control study. PLoS ONE 14(8): e0220858. Moyamoya disease (MMD) is a progressive cerebrovascular disease with unknown etiology. https://doi.org/10.1371/journal.pone.0220858 Growing evidence suggest its involvement of autoimmune and genetic mechanisms in the Editor: Gualtiero I. Colombo, Centro Cardiologico pathogenesis of MMD. This study aims to clarify the association between HLA allele and Monzino, ITALY MMD. Received: February 17, 2019 Accepted: July 24, 2019 Methods Published: August 14, 2019 Case-control study: the DNA of 136 MMD patients in Japan was extracted and the genotype Copyright:© 2019 Tashiro et al. This is an open of human leukocyte antigen (HLA) from this DNA was determined by super-high-resolution access article distributed under the terms of the single-molecule sequence-based typing using next-generation sequencing. Next, the fre- Creative Commons Attribution License, which quency of each HLA allele (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA- permits unrestricted use, distribution, and reproduction in any medium, provided the original DPB1) was compared with those in the Japanese control database. In addition, haplotype author and source are credited. estimation was performed using the expectation maximization algorithm. Data Availability Statement: All relevant data are within the paper and its Supporting Information. Results Funding: This work was supported by Ministry of The frequencies of the HLA-DRB1*04:10 allele (4.77% vs. 1.47% in the control group; P = Health, Labour and Welfare (MHLW) Grant −3 1.7× 10 ; odds ratio [OR] = 3.35) and of the HLA-DRB1*04:10–HLA-DQB1*04:02 haplo- Number S17310031, Japan Agency for Medical −3 Research and Development (AMED) Grant Number type (haplotype frequency 4.41% vs. 1.35% in the control group; P = 2.0× 10 ; OR = 3.37) J170001344 and Japan Society for the promotion significantly increased. The frequency of thyroid diseases, such as Graves’ disease and of Science (JSPS) KAKENHI Grant Number Hashimoto thyroiditis, increased in HLA-DRB1*04:10-positive MMD patients compared 17K10815. GenoDive Pharma Inc. provided support in the form of salaries for H. I., but did not with that in HLA-DRB1*04:10-negative MMD patients. PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 1 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease have any additional role in the study design, data Conclusions collection and analysis, decision to publish, or HLA-DRB1*04:10 is a risk allele and HLA-DRB1*04:10–HLA-DQB1*04:02 a risk haplotype preparation of the manuscript. The specific roles of these authors are articulated in the ‘author for MMD. In addition, HLA-DRB1*04:10 is associated with thyroid disease in MMD patients. contributions’ section. Competing interests: All authors have no conflict of interest related to our manuscript. H.I. is an employee of GenoDive Pharma Inc, and has financial supported from the company in the form Introduction of the salary. Other authors received no specific funding from any companies including Genodive Moyamoya disease (MMD) is a chronic, occlusive cerebrovascular disease with unknown Pharma Inc. H.I., played a role in study design and etiology. MMD involves bilateral steno-occlusive changes at the terminal portion of the inter- data analysis. Genodive Pahrma Inc. does not alter nal carotid artery and the formation of an abnormal vascular network at the base of the brain our adherence to PLOS ONE policies on sharing [1]. MMD is a relatively rare disease, as estimated incidence of MMD is 3.1–10.5/100,000 in data and materials. Japanese population [2–3]. Despite its rarity, MMD is one of the leading causes of cerebrovas- cular diseases among children and young adults in Japan and Korea. Thus, it is expected to unveil the pathogenesis of MMD and diagnose MMD before onset of stroke. Although the etiology of MMD remains undetermined, previous studies showed an association between autoimmunity and MMD [4–11], such as the high frequencies of thyroid dysfunction and type 1 diabetes among MMD patients [4–10]. Human leukocyte antigen (HLA) proteins provide peptides to T-cell receptors for the maintenance of self-tolerance and adapted immunity [12–14]. Allelic and haplotypic differ- ences between HLA alleles are associated mainly with human autoimmune diseases [13–15]. However, despite accumulating evidence, it is unclear how genetic variations of HLA alleles lead to a risk of autoimmune diseases. Recent studies have analyzed HLA–peptide–T-cell receptor complexes and demonstrated the autoantigen recognition mechanism [15–17]. Therefore, to understand the autoimmunity mechanism, it is critical to determine HLA alleles susceptible and/or resistant to autoimmune diseases. Several studies have demonstrated the association between MMD and allelic and haplotypic � � differences of HLA, including both HLA class I and class II alleles (e.g., HLA-A 24, HLA-B 54, � � HLA-DRB1 04:05, and HLA-DQB1 04:01) in multiple cohorts of East Asians [18–21], as well as in Europeans [22]. We sought to determine susceptible allele for MMD using next-genera- tion sequence (NGS), since conventional HLA DNA typing methods, including sequence- based typing (SBT) and sequence-specific oligonucleotide (SSO) typing, yield ambiguous results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment [23]. The recently developed super-high-resolution single-molecule sequence- based typing (SS-SBT) method using NGS can determine an HLA allele sequence at the 8-digit level and also overcome the phase ambiguity of earlier typing methods [24]. In this study, we analyzed the association between MMD and HLA alleles using SS-SBT and NGS in Japanese MMD patients. Materials and methods Study design This is a case-control study. MMD patient samples were collected at Tohoku University hospi- tal and Kohnan hospital. We referred HLA database of the University of Tokyo as for healthy control. Based on previous reports demonstrating HLA alleles and autoimmune diseases [13– 15], we calculated the number of samples to be enrolled in this study as more than 130 MMD patients and 300–500 healthy controls, respectively. Written informed consent for participa- tion in the study was obtained from all subjects and (in the case of pediatric patients) from PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 2 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease their guardians. After obtaining written informed consent, we collected blood samples and performed HLA genotyping. Hardy-Weinberg equation (HWE) was not considered in this study, since previous reports demonstrated deviation from HWE for major histocompatibility region on chromosome 6 [25]. The study complied with the Helsinki Declaration on ethical principles for medical research involving human subjects. Ethical approval was obtained from the ethical committees of Tohoku University, Kohnan Hospital, and the University of Tokyo. Subject population All MMD patients enrolled in the study (n = 136, aged 11–78 years, mean 43.1) met the diag- nostic criteria established by the Research Committee on Spontaneous Occlusion of the Circle of Willis, the Ministry of Health, Labor and Welfare, Japan [26]. Patients with quasi- moya- moya disease were not enrolled in this study. Samples were collected between 2016 and 2018, and most of the patients were residents in Tohoku region in Japan. Clinical characteristics of enrolled patients were extracted from the databases in Tohoku University Hospital and Koh- nan hospital. HLA genotyping We performed HLA genotyping with SS-SBT at the 8-digit level using NGS, as reported in pre- vious studies [24]. Outline of procedures is shown in https://dx.doi.org/10.17504/protocols.io. 3rxgm7n [PROTOCOL DOI]. Briefly, genomic DNA was obtained from the patients’ 2ml of whole blood using the QIAamp DNA Mini Kit for genomic DNA purification (Qiagen GmbH, Hilden, Germany), and 400 ng of purified genomic DNA was used for polymerase chain reaction (PCR) amplification. DNA was preserved in 4˚C freezer. The basic cycling para- meters were as follows: (i) first denaturation at 94˚C for 2 min, followed by 30 cycles of dena- turation at 98˚C for 10 s and 60˚C for 20 s and extension at 68˚C for 5 min (HLA-A, HLA-B, and HLA-C); (ii) first denaturation at 94˚C for 2 min, followed by 30 cycles of denaturation at 98˚C for 10 s and annealing at 70˚C for 5 min (HLA-DRB1 and HLA-DPB1); and (iii) first denaturation at 94˚C for 2 min, followed by 30 cycles of denaturation at 98˚C for 10 s and annealing at 70˚C for 9 min (HLA-DQB1). Long-range PCR reactions were performed using the thermal cycler Gene Amp PCR System 9700 (Life Technologies, Carlsbad, CA, USA). The PCR products obtained were purified with Agencourt AMPure XP (Beckman Coutler, CA, USA) and quantified by the Quant-iT Picogreen dsDNA Assay Kit (Thermo Fisher Scientific, MA, USA). Next, the PCR products were clonally amplified and barcoded using the Ion Plus Fragment Library Kit (Life Technologies), and the barcoded library was sequenced using the Ion Torrent Personal Genome Machine DNA sequencing system (Life Technologies). The NGS read data were analyzed by Sequence Alignment Based Assigning Software (SeaBass), and finally, the HLA alleles were determined. Control database We referred HLA database of the University of Tokyo. Control group does not include patients with MMD and autoimmune disease. Control samples have been collected between 1990 and 2018, and most of the healthy controls were residents in Kanto district in Japan. HLA genotyp- ing was performed, as reported previously [27–28]. HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1, HLA-DQB1, and HLA-DPB1) genotypes were determined with the HLA-DNA Typing Kit, Luminex multianalyte profiling system xMAP (Liminex Corp., Austin, TX, USA), WAKFlow HLA Typing Kit (Wakunaga Pharmaceutical, Osaka, Japan), and LAB- Type SSO HLA Kit (One Lambda, Canoga Park, CA, USA) according to the manufacturers’ instructions. All HLA alleles were determined at the 4-digit level. PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 3 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease Haplotype estimation We performed 2-locus (HLA-DRB1–HLA-DQB1 and HLA-A–HLA-B) and 3-locus (HLA-DR1–HLA-DQB1–HLA-DPB1 and HLA-A–HLA-B–HLA-C) haplotype analysis using Bridging ImmunoGenomic Data-Analysis Workflow Gaps (BigDAWG) [29]. The pairwise linkage disequilibrium (LD) parameters, r and D’, between alleles at different class II HLA loci were calculated on the basis of the haplotype frequencies estimated by the expectation maximi- zation (EM) algorithm [30–31]. Determination of polymorphisms in the RNF213 gene Polymorphisms in the RNF213 gene was determined as previously reported [32]. Single nucleotide polymorphism (SNP) for rs112735431 (https://www.ncbi.nlm.nih.gov/projects/ SNP/snp_ref.cgi?rs=112735431) was analyzed using the TaqMan SNP genotyping assay (Assay ID: C_153120198_10; Applied Biosystems, Foster City, CA, USA) on a StepOnePlus real-time polymerase chain reaction (PCR) system (Applied Biosystems, Foster City, CA, USA). The basic cycling parameters were as follows: hold at 95˚C for 10 minutes, followed by 40 cycles of PCR amplification comprising of denaturation at 95˚C for 15 seconds, and annealing and extension at 60˚C for 1 minute. Genotype calls were evaluated with Applied Biosystems Taq- Man Genotype software. The investigators who assessed genotype were blinded to phenotypic information. Statistical analysis Statistical tests were performed using JMP Pro version 13 (SAS Institute). For each testing sce- nario, prior to statistical analysis, rare alleles (expected counts < 5) were combined into the “Others” category. The differences between allele and haplotype frequencies were calculated by the chi-square test. Adjustment for multiple comparisons was conducted using the Bonfer- roni method. Corrected P-values (P ) were calculated by multiplying the P-values with the number of examined alleles. The association between HLA-DRB1 04:10 and clinical character- istics was evaluated using Mann–Whitney’s U test, and assessment of continuous variables and categorical variables was performed using Fisher’s exact test the chi-square test, respec- tively. Quantitative variables, such as Suzuki stage and age at onset, were stratified into several categories. For example, age at onset was stratified into three categorical variables, eg, child- onset, elderly-onset (age > 60), perinatal period-onset. Multivariate logistic analysis using Poisson regression models was used to adjust for age and sex. P-values of <0.05 were consid- ered statistically significant. Compliance with STrengthening the REporting of Genetic Association Studies (STREGA) Guidelines This study complied with STrengthening the REporting of Genetic Association Studies (STREGA) Guidelines [33], as shown in S1 Table. Results HLA-DRB1 04:10 is a risk allele for MMD In this study, we examined the frequencies of HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1, HLA-DQB1, and HLA-DPB1) alleles in MMD patients and controls. Tables 1–3 shows the frequencies for HLA class II alleles in MMD patients and controls. As seen in the table, HLA-DRB1 04:10 was significantly associated with MMD: allele frequency PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 4 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease Table 1. Frequencies of HLA-DRB1 allele carrier in MMD patients and controls. Locus Allele Control Patient OR (95%CI) P value P (2n = 814) (2n = 272) DRB1 1:01 7.00 (57) 5.88 (16) 0.83 (0.44–1.50) 0.523 NS DRB1 4:03 2.95 (24) 2.57 (7) 0.87 (0.31–2.11) 0.748 NS DRB1 4:05 14.4 (117) 10.3 (28) 0.68 (0.42–1.07) 0.087 NS DRB1 4:06 3.44 (28) 1.10 (3) 0.31 (0.06–1.03) 0.045 NS −3 DRB1 4:10 1.47 (12) 4.77 (13) 3.35 (1.39–8.14) 1.7 x 10 0.030 DRB1 8:02 3.81 (31) 5.15 (14) 1.37 (0.66–2.70) 0.338 NS DRB1 8:03 7.62 (62) 8.09 (22) 1.07 (0.61–1.80) 0.801 NS DRB1 9:01 14.9 (121) 18.0 (49) 1.26 (0.85–1.83) 0.216 NS DRB1 11:01 2.83 (23) 1.84 (5) 0.64 (0.19–0.76) 0.374 NS DRB1 12:01 3.56 (29) 3.31 (9) 0.93 (0.38–2.04) 0.844 NS DRB1 12:02 2.21 (18) 1.47 (4) 0.66 (0.16–2.03) 0.453 NS DRB1 13:02 7.62 (62) 6.25 (17) 0.81 (0.43–1.43) 0.452 NS DRB1 14:05 1.97 (16) 1.84 (5) 0.93 (0.27–2.70) 0.895 NS DRB1 14:06 1.60 (13) 2.94 (8) 1.87 (0.66–4.92) 0.163 NS DRB1 14:54 3.19 (26) 1.47 (4) 0.45 (0.11–1.32) 0.133 NS DRB1 15:01 8.11 (66) 6.25 (17) 0.76 (0.41–1.33) 0.318 NS DRB1 15:02 8.60 (70) 10.3 (28) 1.22 (0.74–1.97) 0.398 NS DRB1 others 4.79 (39) 8.46 (23) 1.84 (1.02–3.22) 0.024 NS The number of allele carriers is shown in parentheses. The association was examined by the chi-square test. Corrected P-values (P ) statistically significant after Bonferroni correction are indicated in bold. OR, odds ratio; CI, confidence interval; P , corrected P-value; NS, not significant. https://doi.org/10.1371/journal.pone.0220858.t001 −3 4.77% vs. 1.47% in the control group; P = 1.7 × 10 ; odds ratio [OR] = 3.35. The frequency of HLA-DRB1 04:06 decreased in MMD patients compared with that in the control group: allele frequency 1.10% vs. 3.44% in the control group; P = 0.045; OR = 0.31. However, after Bonfer- roni correction, the P-value (P ) did not reach the threshold (Table 1). In addition, we found no association between other HLA class II alleles (HLA-DQB1 and HLA-DPB1) and MMD Table 2. Frequencies of HLA-DQB1 allele carrier in MMD patients and controls. locus Allele Control Patient OR (95%CI) P value P (2n = 814) (2n = 272) DQB1 3:01 12.0 (98) 14.3 (39) 1.22 (0.80–1.85) 0.323 NS DQB1 3:02 9.34 (76) 9.19 (25) 0.98 (0.59–1.60) 0.943 NS DQB1 3:03 15.6 (127) 18.4 (50) 1.22 (0.83–1.77) 0.282 NS DQB1 4:01 14.4 (117) 9.56 (26) 0.63 (0.39–1.00) 0.042 NS DQB1 4:02 3.19 (26) 6.99 (19) 2.28 (1.17–4.35) 0.006 NS DQB1 5:01 7.74 (63) 6.62 (18) 0.84 (0.46–1.48) 0.542 NS DQB1 5:02 2.09 (17) 1.47 (4) 0.70 (0.17–2.17) 0.522 NS DQB1 5:03 3.56 (29) 2.94 (8) 0.82 (0.32–1.87) 0.625 NS DQB1 6:01 16.2 (132) 18.0 (49) 1.14 (0.77–1.65) 0.491 NS DQB1 6:02 7.86 (64) 5.88 (16) 0.73 (0.39–1.31) 0.279 NS DQB1 6:04 7.37 (60) 6.25 (17) 0.84 (0.45–1.49) 0.533 NS DQB1 others 0.62 (5) 0.37 (1) 0.60 (0.01–5.37) 0.635 NS The number of allele carriers is shown in parentheses. The association was examined by the chi-square test. Corrected P-values (P ) statistically significant after Bonferroni correction are indicated in bold. OR, odds ratio; CI, confidence interval; P , corrected P-value; NS, not significant. https://doi.org/10.1371/journal.pone.0220858.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 5 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease Table 3. Frequencies of HLA-DPB1 allele carrier in MMD patients and controls. locus Allele Control Patient OR (95%CI) P value P (2n = 814) (2n = 272) DPB1 2:01 25.2 (205) 29.4 (80) 1.24 (0.90–1.69) 0.170 NS DPB1 2:02 4.30 (35) 1.84 (5) 0.42 (0.13–1.08) 0.062 NS DPB1 3:01 4.42 (36) 4.41 (12) 1.00 (0.42–2.00) 0.994 NS DPB1 4:01 6.02 (49) 4.41 (12) 0.72 (0.34–1.40) 0.319 NS DPB1 4:02 10.1 (82) 8.09 (22) 0.79 (0.46–1.30) 0.335 NS DPB1 5:01 38.0 (309) 36.4 (99) 0.94 (0.70–1.25) 0.645 NS DPB1 9:01 7.86 (64) 9.93 (27) 1.29 (0.77–2.11) 0.287 NS DPB1 others 4.18 (34) 5.52 (15) 1.34 (0.67–2.57) 0.357 NS The number of allele carriers is shown in parentheses. The association was examined by the chi-square test. Corrected P-values (P ) statistically significant after Bonferroni correction are indicated in bold. OR, odds ratio; CI, confidence interval; P , corrected P-value; NS, not significant. https://doi.org/10.1371/journal.pone.0220858.t003 (Tables 2 and 3) and no association between HLA class I alleles (HLA-A, HLA-B, and HLA-C) and MMD (S2 Table). Association between the HLA-DR1–HLA-DQB1 haplotype and MMD Table 4 and S2 Table show the haplotype frequency estimated using the EM algorithm. The � � frequency of HLA-DRB1 04:10–HLA-DQB1 04:02 increased in MMD patients (Table 4): hap- −3 lotype frequency 4.41% vs. 1.35% in the control group; P = 2.0 × 10 ; OR = 3.37. On the other � � hand, the frequencies of HLA-DRB1 04:05–HLA-DQB1 04:01 (haplotype frequency 9.56% vs. Table 4. Frequencies of estimated HLA-DRB1-DQB1 haplotype carrier in MMD patients and controls. DRB1/DQB1 Control Patient OR (95%CI) P value P (2n = 814) (2n = 272) � � 01:01- 05:01 7.00 (57) 5.88 (16) 0.83 (0.44–1.50) 0.523 NS � � 04:03- 03:02 2.83 (23) 2.57 (7) 0.91 (0.33–2.22) 0.826 NS � � 04:05- 04:01 14.3 (116) 9.56 (26) 0.64 (0.39–1.01) 0.047 NS � � 04:06- 03:02 3.44 (28) 1.10 (3) 0.31 (0.06–1.03) 0.045 NS � � 04:10- 04:02 1.35 (11) 4.41 (12) 3.37 (1.34–8.53) 0.002 0.036 � � 08:02- 03:02 1.97 (16) 2.94 (8) 1.51 (0.55–3.79) 0.343 NS � � 08:02- 04:02 1.84 (15) 2.21 (6) 1.20 (0.38–3.32) 0.707 NS � � 08:03- 06:01 7.62 (62) 7.72 (21) 1.01 (0.58–1.73) 0.955 NS � � 09:01- 03:03 14.4 (117) 17.3 (47) 1.24 (0.84–1.82) 0.247 NS � � 11:01- 03:01 2.58 (21) 1.84 (5) 0.71 (0.21–1.95) 0.488 NS � � 12:01- 03:01 2.58 (21) 2.94 (8) 1.14 (0.43–2.73) 0.749 NS � � 12:02- 03:01 2.21 (18) 1.47 (4) 0.66 (0.16–2.03) 0.453 NS � � 13:02- 06:04 7.37 (60) 6.25 (17) 0.84 (0.45–1.49) 0.533 NS � � 14:05- 05:03 1.84 (15) 1.84 (5) 1.00 (0.28–2.92) 0.996 NS � � 14:06- 03:01 1.60 (13) 2.94 (8) 1.87 (0.66–4.92) 0.163 NS � � 15:01- 06:02 7.86 (64) 5.88 (16) 0.73 (0.39–1.31) 0.279 NS � � 15:02- 06:01 8.60 (70) 10.3 (28) 1.22 (0.74–1.97) 0.398 NS Others 10.7 (87) 12.9 (35) 1.23 (0.79–1.90) 0.324 NS The number of haplotype carriers estimated by the EM algorithm is shown in parentheses. The association was examined by the chi-square test. Corrected P-values (P ) statistically significant after Bonferroni correction are indicated in bold. , Separator; OR, odds ratio; CI, confidence interval; P , corrected P-value; NS, not significant. https://doi.org/10.1371/journal.pone.0220858.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 6 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease � � 14.3% in the control group; P = 0.047; OR = 0.64) and HLA-DRB1 04:06–HLA-DQB1 03:02 (haplotype frequency 1.10% vs. 3.44% in the control group; P = 0.045; OR = 0.31) decreased in MMD patients. However, after Bonferroni correction, the P-value (P ) did not reach the threshold (Table 4). In addition, we found no association between HLA-DRB1–HLA-DQB1– HLA-DPB1, HLA-A–HLA-B, and HLA-A–HLA-B–HLA-C haplotypes and MMD (S3 Table). High frequency of thyroid dysfunction in HLA-DRB1 04:10-positive MMD patients Table 5 shows the results of our analysis of the association between HLA-DRB1 04:10 and MMD clinical characteristics. The results indicated that the frequency of thyroid diseases, including Graves’ disease (GD) and Hashimoto thyroiditis (HT), is high in HLA-DRB1 04:10- positive MMD patients compared with that in HLA-DRB1 04:10-negative MMD patients (frequency 23.1% vs. 4.1% in the control group; P = 0.029). In addition, the HLA-DRB1 04:10- Table 5. Comparison of demographics between MMD patients with or without HLA-DRB1 04:10. unadjusted Adjusted � � DRB1 04:10 DRB1 04:10 OR (95%CI) P value OR P value -positive -negative (95%CI) (n = 13) (n = 123) Age 38.8 ± 10.8 43.6 ± 15.4 0.258† Sex (Male: Female) 1:12 30:93 0.26 (0.03–2.07) 0.297† History Diabetes 0% (0) 0.8% (1) 0 0.744†ab Thyroid diseases 23.1% (3) 4.1% (5) 7.08 (1.47–34.0) 0.029† 5.61 (1.09–28.9) 0.039 Other autoimmune diseases 0% (0) 0% (0) 0 1.000† Polymorphism c.14576G>A 0.784§ in RNF213 gene G/G 38.5% (5) 30.1% (38) 1.40 (0.43–4.55) 0.549‡ G/A 61.5% (8) 67.4% (83) 0.77 (0.24–2.51) 0.771‡ A/A 0% (0) 1.6% (2) 0 1.000‡ Suzuki stage 0.187§ 1–2 15.4% (2) 27.6% (34) 0.513‡ 3–4 86.4% (11) 60.2% (74) 5–6 0% (0) 12.2% (15) The age of onset 30.1 ± 13.3 35.4 ± 16.0 0.206† Child 15.4% (2) 17.9% (22) 0.48 (0.11–1.75) 0.822‡ Onset >60 0% (0) 4.1% (5) 3.64 (0.77–17.1) 0.459‡ Perinatal period 0% (0) 4.9% (6) 0 0.415‡ Symptom 0.134§ Ischemia 76.9% (10) 88.7% (109) 0.43 (0.11–1.75) 0.428‡ Hemorrhage 23.1% (3) 7.3% (9) 3.80 (0.88–16.3) 0.091‡ Asymptomatic 0% (0) 4.1% (5) 0 0.459‡ The association between MMD patients with or without HLA-DRB1 04:10 was examined using Mann–Whitney’s U test, Fisher’s exact test, or the chi-square test. The number of patients is shown in parentheses, and the percentages are based on the number of patients per category. Mean values are represented as mean ± standard deviation (SD). A multiple logistic regression model was used to adjust for age and sex. † Mann–Whitney’s U test ‡ Fisher’s exact test § chi-square test. RNF213, ring finger protein 213. https://doi.org/10.1371/journal.pone.0220858.t005 PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 7 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease positive group had more females (frequency 92.3% vs. 75.6%; P = 0.297), younger patients (mean age of onset 30.1 ± 13.3 vs. 35.4 ± 16.0 years in the control group; P = 0.206), and higher incidences of hemorrhagic stroke (frequency 23.1% vs. 7.3% in the control group; P = 0.091) than the HLA-DRB1 04:10-negative group. However, these differences were not statistically significant. We also found no association among polymorphisms in the ring finger protein 213 (RNF213) gene, Suzuki stage, and HLA-DRB1 genotype. After adjustment for age and sex, sig- nificantly higher proportion of HLA-DRB1 04:10-positive MMD patients had thyroid disease compared with HLA-DRB1 04:10-negative MMD patients (P = 0.039). Discussion � � In this study, we identified HLA-DRB1 04:10 as a risk allele and HLA-DRB1 04:10–HLA- DQB1 04:02 as a risk haplotype for MMD. This result was inconsistent with previous results, � � � � which demonstrates HLA-DRB1 13:02, DRB1 15:01, DQB1 06:02 and DQB1 06:09, as risk allele for MMD in East Asians [19–21]. This inconsistency might be resulted from lack of rig- orous statistical analysis and limited range of subject population to familial cases in previous studies. Considering Based on the fact that the frequency of HLA-DQB1 04:02, which is in LD with HLA-DRB1 04:10 allele, is not increased compared with the control group, and HLA- � � DRB1 08:02 in LD with HLA-DQB1 04:02 is not increased in MMD patients [34], it is concei- � � vable that HLA-DRB1 04:10, not HLA-DQB1 04:02, is a risk allele for MMD. In addition, � � some HLA-DR-DQ haplotypes, such as HLA-DR9-DQA1 03–HLA-DQB1 03:03 and HLA- � � DR4-DQA1 03–HLA-DQB1 04:01, lead to the risk of type 1 diabetes and autoimmune endo- crinopathies in the Japanese population [13–14]. Therefore, HLA class II alleles are considered to play a key role in autoimmune reactions and it is possible that autoimmune reactions related to HLA-DRB1 04:10 might play a role in MMD pathogenesis, although future studies includ- ing large cohorts should be performed. In this study, we demonstrated that the frequency of thyroid diseases, including GD and HT, is high in HLA-DRB1 04:10-positive MMD patients compared with that in HLA- � � DRB1 04:10-negative MMD patients, suggesting the relationship between the HLA-DRB1 04: 10 haplotype and MMD and autoimmune thyroid diseases. Many studies have reported the association between thyroid dysfunction and MMD [7–12], suggesting that elevated autoanti- body concentrations and hyperthyroidism are associated with stenotic lesions in the terminal portion of the internal carotid artery and aggressive MMD presentations [7–12]. However, the underlying mechanism of this association remains undetermined. Although GD and HT are multifactorial autoimmune diseases [35–38], they show opposite phenotypes: GD is character- ized by the production of thyroid-stimulating hormone receptor–stimulating antibodies, lead- ing to hyperthyroidism, whereas HT is characterized by the apoptosis of thyrocytes, resulting in hypothyroidism. However, GD and HT share an immunological basis; in fact, some alleles, � � � � such as HLA-A 02:07, HLA-B1 35:01, HLA-B1 46:01, and HLA-DRB4 53:01, are shared between GD and HT [35–39]. Although the association of HLA-DRB1 04:10 with thyroid dys- function has never been reported, its association with Vogt–Koyanagi–Harada disease and idiopathic thrombocytopenic purpura is known [40–41]. Future studies are required to clarify the functional role of HLA-DRB1 04:10 and the nature of its involvement in MMD pathogen- esis, especially in patients with thyroid diseases. The role of RNF213 in autoimmune reactions is still unclear. Japanese researchers, includ- ing us, previously demonstrated that RNF213 is a disease susceptibility gene for MMD [42– 44]. The c.14576G>A polymorphism in RNF213 was identified in 95% of familial MMD cases 37,38 and 79% of sporadic MMD cases. The c.14576G>A polymorphism in RNF213 is located in neither the AAA+ATPase domain nor the RING finger ubiquitin ligase domain [42–44]. PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 8 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease Functional roles of RNF213 and its polymorphism in MMD pathogenesis remain undeter- mined. However, since the c.14576G>A polymorphism in RNF213 greatly enhances the risk of MMD pathogenesis and that patients with this polymorphism show earlier disease onset and more severe clinical manifestations than MMD patients without the polymorphism [44], it is conceivable that the c.14576G>A polymorphism in RNF213 plays an important role in MMD pathogenesis. In a previous study, we reported regulatory T-cell decrease in RNF213- knockout mice [45]; therefore, RNF213 might play a role in autoimmune reactions. In con- trast, in this study, we found no association between HLA-DRB1 04:10 and polymorphism in RNF213 and no difference in the soluble CD163 and CXCL5 serum concentrations in MMD patients between the RNF213 variant and normal variant groups [11]. These results suggested that the c.14576G>A polymorphism in RNF213 might not directly affect antigen recognition and the function of antigen-presenting cells. Further research could clarify the involvement of the RNF213 protein in autoimmune reactions. This study had a few limitations. HLA-DRB1 04:10-positive patients account for ~10% of Japanese MMD patients; therefore, its involvement should be investigated in a larger popula- tion or other institutions. In addition, association between thyroid dysfunction and MMD is different between Asians and Caucasians: GD is more prevalent among Japanese MMD patients, whereas HT is more prevalent among Caucasian MMD patients [5–10]. The patients and controls in this study were residents of Tohoku and Kanto districts. Although the distribu- tion of HLA alleles in the Japanese population is relatively homogeneous, there might be a dif- ference in the HLA allele frequencies in MMD patients living in other regions of Japan. Further studies, which includes more control and MMD patients from other institutions, are required to clarify the exact association of HLA-DRB1 04:10 to MMD pathogenesis. Conclusions � � � This study revealed that HLA-DRB1 04:10 and HLA-DRB1 04:10–HLA-DQB1 04:02 are a risk allele and a risk haplotype, respectively, for MMD. In addition, the HLA-DRB1 04:10 fre- quency increases in MMD patients with thyroid diseases. Although further studies are required to clarify the exact association between HLA class II and MMD, autoimmunity might explain MMD pathogenesis, at least in part. Supporting information S1 Table. Strengthening the Reporting of Genetic Association Studies (STREGA) checklist. (DOCX) S2 Table. Frequencies of HLA class I carrier in MMD patients and controls. The number of allele carriers are shown in parentheses. Rare alleles (with expected counts less than five) are combined into “others” category prior to statistical analysis. The association was examined by Chi-square test. The corrected p (P ) values, statistically significant after Bonferroni correction, are indicated in the bold. Abbreviations are as follows; OR, odds ratio; CI, confidence interval; P , corrected p value; NS, not significant. Separator. (DOCX) S3 Table. Frequencies of estimated haplotype carrier in MMD patients and controls. The number of estimated haplotype carriers by the expectation maximization algorithm are shown in parentheses. Rare alleles (with expected counts less than five) are combined into “others” category prior to statistical analysis. The association was examined by Chi-square test. The cor- rected p (P ) values after Bonferroni correction are shown. Abbreviations are as follows; OR, PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 9 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease odds ratio; CI, confidence interval; P , corrected p value; NS, not significant. Separator. (DOCX) Acknowledgments We wish to thank Dr. Jun Yasuda, Dr. Gen Tamiya, Dr. Kazuki Kumada, and Dr. Koyu Ito for their valuable suggestions on study design. We also thank HLA & KIR Imputaion Network (HKimpnet) and Ms. Yuko Okudaira for HLA genotyping by SS-SBT method. Author Contributions Conceptualization: Ryosuke Tashiro, Hiroyuki Sakata. 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Identification of HLA-DRB1*04:10 allele as risk allele for Japanese moyamoya disease and its association with autoimmune thyroid disease: A case-control study

PLoS ONE, Volume 14 (8) – Aug 14, 2019

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Copyright: © 2019 Tashiro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information. Funding: This work was supported by Ministry of Health, Labour and Welfare (MHLW) Grant Number S17310031, Japan Agency for Medical Research and Development (AMED) Grant Number J170001344 and Japan Society for the promotion of Science (JSPS) KAKENHI Grant Number 17K10815. GenoDive Pharma Inc. provided support in the form of salaries for H. I., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: All authors have no conflict of interest related to our manuscript. H.I. is an employee of GenoDive Pharma Inc, and has financial supported from the company in the form of the salary. Other authors received no specific funding from any companies including Genodive Pharma Inc. H.I., played a role in study design and data analysis. Genodive Pahrma Inc. does not alter our adherence to PLOS ONE policies on sharing data and materials.
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Abstract

Tokunaga K, Fujimura M, Sakata H, et al. (2019) Identification of HLA-DRB1 04:10 allele as risk allele for Japanese moyamoya disease and its association with autoimmune thyroid disease: A Background and purpose case-control study. PLoS ONE 14(8): e0220858. Moyamoya disease (MMD) is a progressive cerebrovascular disease with unknown etiology. https://doi.org/10.1371/journal.pone.0220858 Growing evidence suggest its involvement of autoimmune and genetic mechanisms in the Editor: Gualtiero I. Colombo, Centro Cardiologico pathogenesis of MMD. This study aims to clarify the association between HLA allele and Monzino, ITALY MMD. Received: February 17, 2019 Accepted: July 24, 2019 Methods Published: August 14, 2019 Case-control study: the DNA of 136 MMD patients in Japan was extracted and the genotype Copyright:© 2019 Tashiro et al. This is an open of human leukocyte antigen (HLA) from this DNA was determined by super-high-resolution access article distributed under the terms of the single-molecule sequence-based typing using next-generation sequencing. Next, the fre- Creative Commons Attribution License, which quency of each HLA allele (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA- permits unrestricted use, distribution, and reproduction in any medium, provided the original DPB1) was compared with those in the Japanese control database. In addition, haplotype author and source are credited. estimation was performed using the expectation maximization algorithm. Data Availability Statement: All relevant data are within the paper and its Supporting Information. Results Funding: This work was supported by Ministry of The frequencies of the HLA-DRB1*04:10 allele (4.77% vs. 1.47% in the control group; P = Health, Labour and Welfare (MHLW) Grant −3 1.7× 10 ; odds ratio [OR] = 3.35) and of the HLA-DRB1*04:10–HLA-DQB1*04:02 haplo- Number S17310031, Japan Agency for Medical −3 Research and Development (AMED) Grant Number type (haplotype frequency 4.41% vs. 1.35% in the control group; P = 2.0× 10 ; OR = 3.37) J170001344 and Japan Society for the promotion significantly increased. The frequency of thyroid diseases, such as Graves’ disease and of Science (JSPS) KAKENHI Grant Number Hashimoto thyroiditis, increased in HLA-DRB1*04:10-positive MMD patients compared 17K10815. GenoDive Pharma Inc. provided support in the form of salaries for H. I., but did not with that in HLA-DRB1*04:10-negative MMD patients. PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 1 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease have any additional role in the study design, data Conclusions collection and analysis, decision to publish, or HLA-DRB1*04:10 is a risk allele and HLA-DRB1*04:10–HLA-DQB1*04:02 a risk haplotype preparation of the manuscript. The specific roles of these authors are articulated in the ‘author for MMD. In addition, HLA-DRB1*04:10 is associated with thyroid disease in MMD patients. contributions’ section. Competing interests: All authors have no conflict of interest related to our manuscript. H.I. is an employee of GenoDive Pharma Inc, and has financial supported from the company in the form Introduction of the salary. Other authors received no specific funding from any companies including Genodive Moyamoya disease (MMD) is a chronic, occlusive cerebrovascular disease with unknown Pharma Inc. H.I., played a role in study design and etiology. MMD involves bilateral steno-occlusive changes at the terminal portion of the inter- data analysis. Genodive Pahrma Inc. does not alter nal carotid artery and the formation of an abnormal vascular network at the base of the brain our adherence to PLOS ONE policies on sharing [1]. MMD is a relatively rare disease, as estimated incidence of MMD is 3.1–10.5/100,000 in data and materials. Japanese population [2–3]. Despite its rarity, MMD is one of the leading causes of cerebrovas- cular diseases among children and young adults in Japan and Korea. Thus, it is expected to unveil the pathogenesis of MMD and diagnose MMD before onset of stroke. Although the etiology of MMD remains undetermined, previous studies showed an association between autoimmunity and MMD [4–11], such as the high frequencies of thyroid dysfunction and type 1 diabetes among MMD patients [4–10]. Human leukocyte antigen (HLA) proteins provide peptides to T-cell receptors for the maintenance of self-tolerance and adapted immunity [12–14]. Allelic and haplotypic differ- ences between HLA alleles are associated mainly with human autoimmune diseases [13–15]. However, despite accumulating evidence, it is unclear how genetic variations of HLA alleles lead to a risk of autoimmune diseases. Recent studies have analyzed HLA–peptide–T-cell receptor complexes and demonstrated the autoantigen recognition mechanism [15–17]. Therefore, to understand the autoimmunity mechanism, it is critical to determine HLA alleles susceptible and/or resistant to autoimmune diseases. Several studies have demonstrated the association between MMD and allelic and haplotypic � � differences of HLA, including both HLA class I and class II alleles (e.g., HLA-A 24, HLA-B 54, � � HLA-DRB1 04:05, and HLA-DQB1 04:01) in multiple cohorts of East Asians [18–21], as well as in Europeans [22]. We sought to determine susceptible allele for MMD using next-genera- tion sequence (NGS), since conventional HLA DNA typing methods, including sequence- based typing (SBT) and sequence-specific oligonucleotide (SSO) typing, yield ambiguous results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment [23]. The recently developed super-high-resolution single-molecule sequence- based typing (SS-SBT) method using NGS can determine an HLA allele sequence at the 8-digit level and also overcome the phase ambiguity of earlier typing methods [24]. In this study, we analyzed the association between MMD and HLA alleles using SS-SBT and NGS in Japanese MMD patients. Materials and methods Study design This is a case-control study. MMD patient samples were collected at Tohoku University hospi- tal and Kohnan hospital. We referred HLA database of the University of Tokyo as for healthy control. Based on previous reports demonstrating HLA alleles and autoimmune diseases [13– 15], we calculated the number of samples to be enrolled in this study as more than 130 MMD patients and 300–500 healthy controls, respectively. Written informed consent for participa- tion in the study was obtained from all subjects and (in the case of pediatric patients) from PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 2 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease their guardians. After obtaining written informed consent, we collected blood samples and performed HLA genotyping. Hardy-Weinberg equation (HWE) was not considered in this study, since previous reports demonstrated deviation from HWE for major histocompatibility region on chromosome 6 [25]. The study complied with the Helsinki Declaration on ethical principles for medical research involving human subjects. Ethical approval was obtained from the ethical committees of Tohoku University, Kohnan Hospital, and the University of Tokyo. Subject population All MMD patients enrolled in the study (n = 136, aged 11–78 years, mean 43.1) met the diag- nostic criteria established by the Research Committee on Spontaneous Occlusion of the Circle of Willis, the Ministry of Health, Labor and Welfare, Japan [26]. Patients with quasi- moya- moya disease were not enrolled in this study. Samples were collected between 2016 and 2018, and most of the patients were residents in Tohoku region in Japan. Clinical characteristics of enrolled patients were extracted from the databases in Tohoku University Hospital and Koh- nan hospital. HLA genotyping We performed HLA genotyping with SS-SBT at the 8-digit level using NGS, as reported in pre- vious studies [24]. Outline of procedures is shown in https://dx.doi.org/10.17504/protocols.io. 3rxgm7n [PROTOCOL DOI]. Briefly, genomic DNA was obtained from the patients’ 2ml of whole blood using the QIAamp DNA Mini Kit for genomic DNA purification (Qiagen GmbH, Hilden, Germany), and 400 ng of purified genomic DNA was used for polymerase chain reaction (PCR) amplification. DNA was preserved in 4˚C freezer. The basic cycling para- meters were as follows: (i) first denaturation at 94˚C for 2 min, followed by 30 cycles of dena- turation at 98˚C for 10 s and 60˚C for 20 s and extension at 68˚C for 5 min (HLA-A, HLA-B, and HLA-C); (ii) first denaturation at 94˚C for 2 min, followed by 30 cycles of denaturation at 98˚C for 10 s and annealing at 70˚C for 5 min (HLA-DRB1 and HLA-DPB1); and (iii) first denaturation at 94˚C for 2 min, followed by 30 cycles of denaturation at 98˚C for 10 s and annealing at 70˚C for 9 min (HLA-DQB1). Long-range PCR reactions were performed using the thermal cycler Gene Amp PCR System 9700 (Life Technologies, Carlsbad, CA, USA). The PCR products obtained were purified with Agencourt AMPure XP (Beckman Coutler, CA, USA) and quantified by the Quant-iT Picogreen dsDNA Assay Kit (Thermo Fisher Scientific, MA, USA). Next, the PCR products were clonally amplified and barcoded using the Ion Plus Fragment Library Kit (Life Technologies), and the barcoded library was sequenced using the Ion Torrent Personal Genome Machine DNA sequencing system (Life Technologies). The NGS read data were analyzed by Sequence Alignment Based Assigning Software (SeaBass), and finally, the HLA alleles were determined. Control database We referred HLA database of the University of Tokyo. Control group does not include patients with MMD and autoimmune disease. Control samples have been collected between 1990 and 2018, and most of the healthy controls were residents in Kanto district in Japan. HLA genotyp- ing was performed, as reported previously [27–28]. HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1, HLA-DQB1, and HLA-DPB1) genotypes were determined with the HLA-DNA Typing Kit, Luminex multianalyte profiling system xMAP (Liminex Corp., Austin, TX, USA), WAKFlow HLA Typing Kit (Wakunaga Pharmaceutical, Osaka, Japan), and LAB- Type SSO HLA Kit (One Lambda, Canoga Park, CA, USA) according to the manufacturers’ instructions. All HLA alleles were determined at the 4-digit level. PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 3 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease Haplotype estimation We performed 2-locus (HLA-DRB1–HLA-DQB1 and HLA-A–HLA-B) and 3-locus (HLA-DR1–HLA-DQB1–HLA-DPB1 and HLA-A–HLA-B–HLA-C) haplotype analysis using Bridging ImmunoGenomic Data-Analysis Workflow Gaps (BigDAWG) [29]. The pairwise linkage disequilibrium (LD) parameters, r and D’, between alleles at different class II HLA loci were calculated on the basis of the haplotype frequencies estimated by the expectation maximi- zation (EM) algorithm [30–31]. Determination of polymorphisms in the RNF213 gene Polymorphisms in the RNF213 gene was determined as previously reported [32]. Single nucleotide polymorphism (SNP) for rs112735431 (https://www.ncbi.nlm.nih.gov/projects/ SNP/snp_ref.cgi?rs=112735431) was analyzed using the TaqMan SNP genotyping assay (Assay ID: C_153120198_10; Applied Biosystems, Foster City, CA, USA) on a StepOnePlus real-time polymerase chain reaction (PCR) system (Applied Biosystems, Foster City, CA, USA). The basic cycling parameters were as follows: hold at 95˚C for 10 minutes, followed by 40 cycles of PCR amplification comprising of denaturation at 95˚C for 15 seconds, and annealing and extension at 60˚C for 1 minute. Genotype calls were evaluated with Applied Biosystems Taq- Man Genotype software. The investigators who assessed genotype were blinded to phenotypic information. Statistical analysis Statistical tests were performed using JMP Pro version 13 (SAS Institute). For each testing sce- nario, prior to statistical analysis, rare alleles (expected counts < 5) were combined into the “Others” category. The differences between allele and haplotype frequencies were calculated by the chi-square test. Adjustment for multiple comparisons was conducted using the Bonfer- roni method. Corrected P-values (P ) were calculated by multiplying the P-values with the number of examined alleles. The association between HLA-DRB1 04:10 and clinical character- istics was evaluated using Mann–Whitney’s U test, and assessment of continuous variables and categorical variables was performed using Fisher’s exact test the chi-square test, respec- tively. Quantitative variables, such as Suzuki stage and age at onset, were stratified into several categories. For example, age at onset was stratified into three categorical variables, eg, child- onset, elderly-onset (age > 60), perinatal period-onset. Multivariate logistic analysis using Poisson regression models was used to adjust for age and sex. P-values of <0.05 were consid- ered statistically significant. Compliance with STrengthening the REporting of Genetic Association Studies (STREGA) Guidelines This study complied with STrengthening the REporting of Genetic Association Studies (STREGA) Guidelines [33], as shown in S1 Table. Results HLA-DRB1 04:10 is a risk allele for MMD In this study, we examined the frequencies of HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DRB1, HLA-DQB1, and HLA-DPB1) alleles in MMD patients and controls. Tables 1–3 shows the frequencies for HLA class II alleles in MMD patients and controls. As seen in the table, HLA-DRB1 04:10 was significantly associated with MMD: allele frequency PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 4 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease Table 1. Frequencies of HLA-DRB1 allele carrier in MMD patients and controls. Locus Allele Control Patient OR (95%CI) P value P (2n = 814) (2n = 272) DRB1 1:01 7.00 (57) 5.88 (16) 0.83 (0.44–1.50) 0.523 NS DRB1 4:03 2.95 (24) 2.57 (7) 0.87 (0.31–2.11) 0.748 NS DRB1 4:05 14.4 (117) 10.3 (28) 0.68 (0.42–1.07) 0.087 NS DRB1 4:06 3.44 (28) 1.10 (3) 0.31 (0.06–1.03) 0.045 NS −3 DRB1 4:10 1.47 (12) 4.77 (13) 3.35 (1.39–8.14) 1.7 x 10 0.030 DRB1 8:02 3.81 (31) 5.15 (14) 1.37 (0.66–2.70) 0.338 NS DRB1 8:03 7.62 (62) 8.09 (22) 1.07 (0.61–1.80) 0.801 NS DRB1 9:01 14.9 (121) 18.0 (49) 1.26 (0.85–1.83) 0.216 NS DRB1 11:01 2.83 (23) 1.84 (5) 0.64 (0.19–0.76) 0.374 NS DRB1 12:01 3.56 (29) 3.31 (9) 0.93 (0.38–2.04) 0.844 NS DRB1 12:02 2.21 (18) 1.47 (4) 0.66 (0.16–2.03) 0.453 NS DRB1 13:02 7.62 (62) 6.25 (17) 0.81 (0.43–1.43) 0.452 NS DRB1 14:05 1.97 (16) 1.84 (5) 0.93 (0.27–2.70) 0.895 NS DRB1 14:06 1.60 (13) 2.94 (8) 1.87 (0.66–4.92) 0.163 NS DRB1 14:54 3.19 (26) 1.47 (4) 0.45 (0.11–1.32) 0.133 NS DRB1 15:01 8.11 (66) 6.25 (17) 0.76 (0.41–1.33) 0.318 NS DRB1 15:02 8.60 (70) 10.3 (28) 1.22 (0.74–1.97) 0.398 NS DRB1 others 4.79 (39) 8.46 (23) 1.84 (1.02–3.22) 0.024 NS The number of allele carriers is shown in parentheses. The association was examined by the chi-square test. Corrected P-values (P ) statistically significant after Bonferroni correction are indicated in bold. OR, odds ratio; CI, confidence interval; P , corrected P-value; NS, not significant. https://doi.org/10.1371/journal.pone.0220858.t001 −3 4.77% vs. 1.47% in the control group; P = 1.7 × 10 ; odds ratio [OR] = 3.35. The frequency of HLA-DRB1 04:06 decreased in MMD patients compared with that in the control group: allele frequency 1.10% vs. 3.44% in the control group; P = 0.045; OR = 0.31. However, after Bonfer- roni correction, the P-value (P ) did not reach the threshold (Table 1). In addition, we found no association between other HLA class II alleles (HLA-DQB1 and HLA-DPB1) and MMD Table 2. Frequencies of HLA-DQB1 allele carrier in MMD patients and controls. locus Allele Control Patient OR (95%CI) P value P (2n = 814) (2n = 272) DQB1 3:01 12.0 (98) 14.3 (39) 1.22 (0.80–1.85) 0.323 NS DQB1 3:02 9.34 (76) 9.19 (25) 0.98 (0.59–1.60) 0.943 NS DQB1 3:03 15.6 (127) 18.4 (50) 1.22 (0.83–1.77) 0.282 NS DQB1 4:01 14.4 (117) 9.56 (26) 0.63 (0.39–1.00) 0.042 NS DQB1 4:02 3.19 (26) 6.99 (19) 2.28 (1.17–4.35) 0.006 NS DQB1 5:01 7.74 (63) 6.62 (18) 0.84 (0.46–1.48) 0.542 NS DQB1 5:02 2.09 (17) 1.47 (4) 0.70 (0.17–2.17) 0.522 NS DQB1 5:03 3.56 (29) 2.94 (8) 0.82 (0.32–1.87) 0.625 NS DQB1 6:01 16.2 (132) 18.0 (49) 1.14 (0.77–1.65) 0.491 NS DQB1 6:02 7.86 (64) 5.88 (16) 0.73 (0.39–1.31) 0.279 NS DQB1 6:04 7.37 (60) 6.25 (17) 0.84 (0.45–1.49) 0.533 NS DQB1 others 0.62 (5) 0.37 (1) 0.60 (0.01–5.37) 0.635 NS The number of allele carriers is shown in parentheses. The association was examined by the chi-square test. Corrected P-values (P ) statistically significant after Bonferroni correction are indicated in bold. OR, odds ratio; CI, confidence interval; P , corrected P-value; NS, not significant. https://doi.org/10.1371/journal.pone.0220858.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 5 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease Table 3. Frequencies of HLA-DPB1 allele carrier in MMD patients and controls. locus Allele Control Patient OR (95%CI) P value P (2n = 814) (2n = 272) DPB1 2:01 25.2 (205) 29.4 (80) 1.24 (0.90–1.69) 0.170 NS DPB1 2:02 4.30 (35) 1.84 (5) 0.42 (0.13–1.08) 0.062 NS DPB1 3:01 4.42 (36) 4.41 (12) 1.00 (0.42–2.00) 0.994 NS DPB1 4:01 6.02 (49) 4.41 (12) 0.72 (0.34–1.40) 0.319 NS DPB1 4:02 10.1 (82) 8.09 (22) 0.79 (0.46–1.30) 0.335 NS DPB1 5:01 38.0 (309) 36.4 (99) 0.94 (0.70–1.25) 0.645 NS DPB1 9:01 7.86 (64) 9.93 (27) 1.29 (0.77–2.11) 0.287 NS DPB1 others 4.18 (34) 5.52 (15) 1.34 (0.67–2.57) 0.357 NS The number of allele carriers is shown in parentheses. The association was examined by the chi-square test. Corrected P-values (P ) statistically significant after Bonferroni correction are indicated in bold. OR, odds ratio; CI, confidence interval; P , corrected P-value; NS, not significant. https://doi.org/10.1371/journal.pone.0220858.t003 (Tables 2 and 3) and no association between HLA class I alleles (HLA-A, HLA-B, and HLA-C) and MMD (S2 Table). Association between the HLA-DR1–HLA-DQB1 haplotype and MMD Table 4 and S2 Table show the haplotype frequency estimated using the EM algorithm. The � � frequency of HLA-DRB1 04:10–HLA-DQB1 04:02 increased in MMD patients (Table 4): hap- −3 lotype frequency 4.41% vs. 1.35% in the control group; P = 2.0 × 10 ; OR = 3.37. On the other � � hand, the frequencies of HLA-DRB1 04:05–HLA-DQB1 04:01 (haplotype frequency 9.56% vs. Table 4. Frequencies of estimated HLA-DRB1-DQB1 haplotype carrier in MMD patients and controls. DRB1/DQB1 Control Patient OR (95%CI) P value P (2n = 814) (2n = 272) � � 01:01- 05:01 7.00 (57) 5.88 (16) 0.83 (0.44–1.50) 0.523 NS � � 04:03- 03:02 2.83 (23) 2.57 (7) 0.91 (0.33–2.22) 0.826 NS � � 04:05- 04:01 14.3 (116) 9.56 (26) 0.64 (0.39–1.01) 0.047 NS � � 04:06- 03:02 3.44 (28) 1.10 (3) 0.31 (0.06–1.03) 0.045 NS � � 04:10- 04:02 1.35 (11) 4.41 (12) 3.37 (1.34–8.53) 0.002 0.036 � � 08:02- 03:02 1.97 (16) 2.94 (8) 1.51 (0.55–3.79) 0.343 NS � � 08:02- 04:02 1.84 (15) 2.21 (6) 1.20 (0.38–3.32) 0.707 NS � � 08:03- 06:01 7.62 (62) 7.72 (21) 1.01 (0.58–1.73) 0.955 NS � � 09:01- 03:03 14.4 (117) 17.3 (47) 1.24 (0.84–1.82) 0.247 NS � � 11:01- 03:01 2.58 (21) 1.84 (5) 0.71 (0.21–1.95) 0.488 NS � � 12:01- 03:01 2.58 (21) 2.94 (8) 1.14 (0.43–2.73) 0.749 NS � � 12:02- 03:01 2.21 (18) 1.47 (4) 0.66 (0.16–2.03) 0.453 NS � � 13:02- 06:04 7.37 (60) 6.25 (17) 0.84 (0.45–1.49) 0.533 NS � � 14:05- 05:03 1.84 (15) 1.84 (5) 1.00 (0.28–2.92) 0.996 NS � � 14:06- 03:01 1.60 (13) 2.94 (8) 1.87 (0.66–4.92) 0.163 NS � � 15:01- 06:02 7.86 (64) 5.88 (16) 0.73 (0.39–1.31) 0.279 NS � � 15:02- 06:01 8.60 (70) 10.3 (28) 1.22 (0.74–1.97) 0.398 NS Others 10.7 (87) 12.9 (35) 1.23 (0.79–1.90) 0.324 NS The number of haplotype carriers estimated by the EM algorithm is shown in parentheses. The association was examined by the chi-square test. Corrected P-values (P ) statistically significant after Bonferroni correction are indicated in bold. , Separator; OR, odds ratio; CI, confidence interval; P , corrected P-value; NS, not significant. https://doi.org/10.1371/journal.pone.0220858.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 6 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease � � 14.3% in the control group; P = 0.047; OR = 0.64) and HLA-DRB1 04:06–HLA-DQB1 03:02 (haplotype frequency 1.10% vs. 3.44% in the control group; P = 0.045; OR = 0.31) decreased in MMD patients. However, after Bonferroni correction, the P-value (P ) did not reach the threshold (Table 4). In addition, we found no association between HLA-DRB1–HLA-DQB1– HLA-DPB1, HLA-A–HLA-B, and HLA-A–HLA-B–HLA-C haplotypes and MMD (S3 Table). High frequency of thyroid dysfunction in HLA-DRB1 04:10-positive MMD patients Table 5 shows the results of our analysis of the association between HLA-DRB1 04:10 and MMD clinical characteristics. The results indicated that the frequency of thyroid diseases, including Graves’ disease (GD) and Hashimoto thyroiditis (HT), is high in HLA-DRB1 04:10- positive MMD patients compared with that in HLA-DRB1 04:10-negative MMD patients (frequency 23.1% vs. 4.1% in the control group; P = 0.029). In addition, the HLA-DRB1 04:10- Table 5. Comparison of demographics between MMD patients with or without HLA-DRB1 04:10. unadjusted Adjusted � � DRB1 04:10 DRB1 04:10 OR (95%CI) P value OR P value -positive -negative (95%CI) (n = 13) (n = 123) Age 38.8 ± 10.8 43.6 ± 15.4 0.258† Sex (Male: Female) 1:12 30:93 0.26 (0.03–2.07) 0.297† History Diabetes 0% (0) 0.8% (1) 0 0.744†ab Thyroid diseases 23.1% (3) 4.1% (5) 7.08 (1.47–34.0) 0.029† 5.61 (1.09–28.9) 0.039 Other autoimmune diseases 0% (0) 0% (0) 0 1.000† Polymorphism c.14576G>A 0.784§ in RNF213 gene G/G 38.5% (5) 30.1% (38) 1.40 (0.43–4.55) 0.549‡ G/A 61.5% (8) 67.4% (83) 0.77 (0.24–2.51) 0.771‡ A/A 0% (0) 1.6% (2) 0 1.000‡ Suzuki stage 0.187§ 1–2 15.4% (2) 27.6% (34) 0.513‡ 3–4 86.4% (11) 60.2% (74) 5–6 0% (0) 12.2% (15) The age of onset 30.1 ± 13.3 35.4 ± 16.0 0.206† Child 15.4% (2) 17.9% (22) 0.48 (0.11–1.75) 0.822‡ Onset >60 0% (0) 4.1% (5) 3.64 (0.77–17.1) 0.459‡ Perinatal period 0% (0) 4.9% (6) 0 0.415‡ Symptom 0.134§ Ischemia 76.9% (10) 88.7% (109) 0.43 (0.11–1.75) 0.428‡ Hemorrhage 23.1% (3) 7.3% (9) 3.80 (0.88–16.3) 0.091‡ Asymptomatic 0% (0) 4.1% (5) 0 0.459‡ The association between MMD patients with or without HLA-DRB1 04:10 was examined using Mann–Whitney’s U test, Fisher’s exact test, or the chi-square test. The number of patients is shown in parentheses, and the percentages are based on the number of patients per category. Mean values are represented as mean ± standard deviation (SD). A multiple logistic regression model was used to adjust for age and sex. † Mann–Whitney’s U test ‡ Fisher’s exact test § chi-square test. RNF213, ring finger protein 213. https://doi.org/10.1371/journal.pone.0220858.t005 PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 7 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease positive group had more females (frequency 92.3% vs. 75.6%; P = 0.297), younger patients (mean age of onset 30.1 ± 13.3 vs. 35.4 ± 16.0 years in the control group; P = 0.206), and higher incidences of hemorrhagic stroke (frequency 23.1% vs. 7.3% in the control group; P = 0.091) than the HLA-DRB1 04:10-negative group. However, these differences were not statistically significant. We also found no association among polymorphisms in the ring finger protein 213 (RNF213) gene, Suzuki stage, and HLA-DRB1 genotype. After adjustment for age and sex, sig- nificantly higher proportion of HLA-DRB1 04:10-positive MMD patients had thyroid disease compared with HLA-DRB1 04:10-negative MMD patients (P = 0.039). Discussion � � In this study, we identified HLA-DRB1 04:10 as a risk allele and HLA-DRB1 04:10–HLA- DQB1 04:02 as a risk haplotype for MMD. This result was inconsistent with previous results, � � � � which demonstrates HLA-DRB1 13:02, DRB1 15:01, DQB1 06:02 and DQB1 06:09, as risk allele for MMD in East Asians [19–21]. This inconsistency might be resulted from lack of rig- orous statistical analysis and limited range of subject population to familial cases in previous studies. Considering Based on the fact that the frequency of HLA-DQB1 04:02, which is in LD with HLA-DRB1 04:10 allele, is not increased compared with the control group, and HLA- � � DRB1 08:02 in LD with HLA-DQB1 04:02 is not increased in MMD patients [34], it is concei- � � vable that HLA-DRB1 04:10, not HLA-DQB1 04:02, is a risk allele for MMD. In addition, � � some HLA-DR-DQ haplotypes, such as HLA-DR9-DQA1 03–HLA-DQB1 03:03 and HLA- � � DR4-DQA1 03–HLA-DQB1 04:01, lead to the risk of type 1 diabetes and autoimmune endo- crinopathies in the Japanese population [13–14]. Therefore, HLA class II alleles are considered to play a key role in autoimmune reactions and it is possible that autoimmune reactions related to HLA-DRB1 04:10 might play a role in MMD pathogenesis, although future studies includ- ing large cohorts should be performed. In this study, we demonstrated that the frequency of thyroid diseases, including GD and HT, is high in HLA-DRB1 04:10-positive MMD patients compared with that in HLA- � � DRB1 04:10-negative MMD patients, suggesting the relationship between the HLA-DRB1 04: 10 haplotype and MMD and autoimmune thyroid diseases. Many studies have reported the association between thyroid dysfunction and MMD [7–12], suggesting that elevated autoanti- body concentrations and hyperthyroidism are associated with stenotic lesions in the terminal portion of the internal carotid artery and aggressive MMD presentations [7–12]. However, the underlying mechanism of this association remains undetermined. Although GD and HT are multifactorial autoimmune diseases [35–38], they show opposite phenotypes: GD is character- ized by the production of thyroid-stimulating hormone receptor–stimulating antibodies, lead- ing to hyperthyroidism, whereas HT is characterized by the apoptosis of thyrocytes, resulting in hypothyroidism. However, GD and HT share an immunological basis; in fact, some alleles, � � � � such as HLA-A 02:07, HLA-B1 35:01, HLA-B1 46:01, and HLA-DRB4 53:01, are shared between GD and HT [35–39]. Although the association of HLA-DRB1 04:10 with thyroid dys- function has never been reported, its association with Vogt–Koyanagi–Harada disease and idiopathic thrombocytopenic purpura is known [40–41]. Future studies are required to clarify the functional role of HLA-DRB1 04:10 and the nature of its involvement in MMD pathogen- esis, especially in patients with thyroid diseases. The role of RNF213 in autoimmune reactions is still unclear. Japanese researchers, includ- ing us, previously demonstrated that RNF213 is a disease susceptibility gene for MMD [42– 44]. The c.14576G>A polymorphism in RNF213 was identified in 95% of familial MMD cases 37,38 and 79% of sporadic MMD cases. The c.14576G>A polymorphism in RNF213 is located in neither the AAA+ATPase domain nor the RING finger ubiquitin ligase domain [42–44]. PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 8 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease Functional roles of RNF213 and its polymorphism in MMD pathogenesis remain undeter- mined. However, since the c.14576G>A polymorphism in RNF213 greatly enhances the risk of MMD pathogenesis and that patients with this polymorphism show earlier disease onset and more severe clinical manifestations than MMD patients without the polymorphism [44], it is conceivable that the c.14576G>A polymorphism in RNF213 plays an important role in MMD pathogenesis. In a previous study, we reported regulatory T-cell decrease in RNF213- knockout mice [45]; therefore, RNF213 might play a role in autoimmune reactions. In con- trast, in this study, we found no association between HLA-DRB1 04:10 and polymorphism in RNF213 and no difference in the soluble CD163 and CXCL5 serum concentrations in MMD patients between the RNF213 variant and normal variant groups [11]. These results suggested that the c.14576G>A polymorphism in RNF213 might not directly affect antigen recognition and the function of antigen-presenting cells. Further research could clarify the involvement of the RNF213 protein in autoimmune reactions. This study had a few limitations. HLA-DRB1 04:10-positive patients account for ~10% of Japanese MMD patients; therefore, its involvement should be investigated in a larger popula- tion or other institutions. In addition, association between thyroid dysfunction and MMD is different between Asians and Caucasians: GD is more prevalent among Japanese MMD patients, whereas HT is more prevalent among Caucasian MMD patients [5–10]. The patients and controls in this study were residents of Tohoku and Kanto districts. Although the distribu- tion of HLA alleles in the Japanese population is relatively homogeneous, there might be a dif- ference in the HLA allele frequencies in MMD patients living in other regions of Japan. Further studies, which includes more control and MMD patients from other institutions, are required to clarify the exact association of HLA-DRB1 04:10 to MMD pathogenesis. Conclusions � � � This study revealed that HLA-DRB1 04:10 and HLA-DRB1 04:10–HLA-DQB1 04:02 are a risk allele and a risk haplotype, respectively, for MMD. In addition, the HLA-DRB1 04:10 fre- quency increases in MMD patients with thyroid diseases. Although further studies are required to clarify the exact association between HLA class II and MMD, autoimmunity might explain MMD pathogenesis, at least in part. Supporting information S1 Table. Strengthening the Reporting of Genetic Association Studies (STREGA) checklist. (DOCX) S2 Table. Frequencies of HLA class I carrier in MMD patients and controls. The number of allele carriers are shown in parentheses. Rare alleles (with expected counts less than five) are combined into “others” category prior to statistical analysis. The association was examined by Chi-square test. The corrected p (P ) values, statistically significant after Bonferroni correction, are indicated in the bold. Abbreviations are as follows; OR, odds ratio; CI, confidence interval; P , corrected p value; NS, not significant. Separator. (DOCX) S3 Table. Frequencies of estimated haplotype carrier in MMD patients and controls. The number of estimated haplotype carriers by the expectation maximization algorithm are shown in parentheses. Rare alleles (with expected counts less than five) are combined into “others” category prior to statistical analysis. The association was examined by Chi-square test. The cor- rected p (P ) values after Bonferroni correction are shown. Abbreviations are as follows; OR, PLOS ONE | https://doi.org/10.1371/journal.pone.0220858 August 14, 2019 9 / 12 HLA-DRB1*04:10 is a risk allele for moyamoya disease odds ratio; CI, confidence interval; P , corrected p value; NS, not significant. Separator. (DOCX) Acknowledgments We wish to thank Dr. Jun Yasuda, Dr. Gen Tamiya, Dr. Kazuki Kumada, and Dr. Koyu Ito for their valuable suggestions on study design. We also thank HLA & KIR Imputaion Network (HKimpnet) and Ms. Yuko Okudaira for HLA genotyping by SS-SBT method. Author Contributions Conceptualization: Ryosuke Tashiro, Hiroyuki Sakata. 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