The Clinical Characteristics of Other HLA-B Types in Chinese Ankylosing Spondylitis Patients

HLA-B27 has an established relationship with the development of ankylosing spondylitis (AS). After reviewing the HLA-B genotype from 407 Chinese subjects (318 patients and 89 sex-matched controls), we found that 252 patients and 32 controls were HLA-B27(+) and that HLA-B*27:04 was the dominant HLA-B27 subtype (N = 224). In all participants, HLA*27:04 homozygous were only detected in two patients. In the HLA-B27(+) group, HLA-B40 was observed in 51 cases and one control (p < 0.05, OR = 7.87, 95% CI 1.05–59.0); of these, the most genotype was HLA-B*27:04/B*40:01(N = 38). Two hundred thirty-nine patients' clinical information was recorded. Cases with HLA-B27/B46 had more peripheral joint involvement (OR = 3.95, 95% CI 1.77–8.79) in HLA-B27(+) AS. HLA-B*15:02 may be a significant risk element to peripheral joint involvement (p < 0.05) in HLA-B27(−) patients. Therefore, we believe HLA-B*40:01, HLA-B*46:01, and HLA-B*15:02 can be the test indicators for AS diagnostic value.


INTRODUCTION
Human leukocyte antigen (HLA)-B27 is the most critical gene in ankylosing spondylitis (AS). About 90-95% of AS cases were HLA-B27 positive, while only 1-2% of HLA-B27 positive persons can develop to AS (1,2). Results showed that the occurrence of AS with HLA-B27 appeared in family aggregation. Among the first-degree relatives of HLA-B27 positive AS, the prevalence is 10-30% (3). Above 45 HLA-B27 subtypes, like B * 27:02, B * 27:10, and B * 27:15, were found to be associated with AS, and their distribution varied in different populations (4, 5). B * 27:04 is the primary subtype in the Chinese Han population (6), whereas the Caucasian people are dominated by the B * 27:05 (4). On the contrary, B * 27:06 and B * 27:09 are unrelated to AS. Previous research found homozygous B * 27:04 can affect AS susceptibility but not its clinical manifestations and functional disability (7,8). How about HLA-B27 heterozygote with other HLA-B alleles in AS? Our studies aimed to evaluate the influence of heterozygous HLA-B27 on the clinical manifestations of AS patients.

Study Subjects
Three hundred eighteen Chinese Han patients and 89 sexmatched controls were recruited from the hospitals in Guangdong Province of China. All patients were older than 18 years old and met the 1984 modified New York criteria for AS (9). Two hundred thirty-nine patients had their clinical information collected by two trained rheumatologists during a face-to-face interview at the study visit. Clinical information included peripheral manifestations (uveitis, peripheral joint involvement, dactylitis, and enthesitis), onset age, body mass index (BMI), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and Bath Ankylosing Spondylitis Functional Index (BASFI). We also collected past and current medications, including non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, diseasemodifying antirheumatic drugs (DMARDs), and biologic agents. The general information included age, gender, and smoking (current) and drinking history (current). According to the 2009 ASAS classification criteria (either axial or peripheral) (10), patients without any peripheral manifestations (uveitis, peripheral joint involvement, dactylitis, and enthesitis) were classified as the axial AS (axAS). Controls were free of any history of rheumatic disease. Written informed consent was obtained from all the subjects. The ethics committee of the Third Affiliated Hospital of Sun Yat-Sen University approved our study. All participants gave written informed consent before enrollment.

HLA-B Genotype
Genomic DNA was extracted from peripheral blood using a standard salting-out method. All of the individuals were genotyped for HLA-B loci using the polymerase chain reaction sequence-based typing (PCRS-BT) method. Briefly, we performed locus-specific PCR amplification and bidirectional Sanger sequencing of HLA-B exons 2, 3, and 4. Amplification and sequencing of relevant exons was performed using "in-house" primers. Sequencing was performed on a 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA). The typing results were accomplished using uTYPE v6.0 software (One Lambda, Canoga Park, CA, USA) against the IMGT/HLA database. When encountering ambiguous genotyping results (several genotypic combinations perform identically on sequencing results), alleles were assigned by referring to the most common alleles in the Chinese population (11).

Statistical Analysis
We analyzed the data in two steps. In step 1, we analyzed the HLA-B types in all participants. Then we researched the relationship with HLA-B types and clinical phenotype (Figure 1). For continuous variables, we calculated mean ± standard deviation (SD) and percentage for categorical variables. We performed Student's t-test or rank-sum test to make group

RESULTS
Step 1 The HLA-B Genotypes Distribution in all Samples A total of 407 subjects were analyzed using HLA-B typing including 318 AS and 89 sex-matched controls (Figure 1) from January 2016 to September 2020. As shown in  Figure 2.
In nine B27(−) patients with peripheral joint involvement patients, seven cases (77.78%) carried HLA-B15. There was a significant difference in HLA-B15 (p = 0.014) ( Table 6). For a small number of HLA-B27(−) patients with other manifestations (uveitis and enthesitis), the HLA-B type distributions are shown in Figure 6. One patient with dactylitis has a HLA-B46/B58 in his HLA-B allele.

The Clinical Manifestations in HLA-B27 Subtypes Homozygote and Heterozygote AS Patients
The most frequent subtype was HLA-B * 27:04 in the B27(+) group, so we further analyzed the clinical manifestations of HLA-B * 27:04 homozygous and heterozygous AS patients. One of the HLA-B * 27:04/B * 27:04 patients was a woman who only had the axial phenotype. An X-ray showed that the front edge of each vertebra of the spine was straight, the facet joints of the thoracolumbar and lumbar vertebra were blurred, that there was bony ankylosing, scoliosis, atlantoaxial subluxation, bilateral sacroiliac joint fusion, narrowed hip space, osteoid destruction, and that the surface of the left ischial tuberosity was rough.
In  Table 4). Other HLA-B27 subtypes were also heterozygous, and their clinical peripheral phenotypes are shown in Table 3.   patients had a higher frequency of extra-spine manifestations (12). Research about Korean AS patients found that HLA-B27 homozygosity has no significant difference with heterozygosity on the clinical manifestations and radiographic progression (7,8). Some research found only four homozygous of B * 27:04 in 245 HLA-B27-positive AS patients (13). In our study, we found two HLA-B  (14). HLA-B40 can increase HLA-B27 susceptibility to AS (15,16). The different subtypes had different peripheral manifestations.
HLA-B46 can increase the risk of severe sacroiliitis development related to Japanese psoriatic arthritis (PsA) patients (17). The HLA-A2-B46-DR8 haplotype has a relationship with the levels of complement components (18). HLA-B * 46:01 was the only subtype of HLA-B46 found in our data. In HLA-B27 AS, the frequency was second to that of HLA-B40. Relative to other HLA-B alleles, patients with HLA-B * 27:04/B * 46:01 had a higher prevalence of peripheral joint involvement. HLA-B * 46:01 was associated with peripheral joint involvement in HLA-B * 27:04 AS patients.
Seventeen B27(+) patients showed B58. There was no difference between patients and controls. As we all know, Allopurinol-induced severe cutaneous adverse drug reactions (SCAR) is strongly associated with the presence of HLA-B * 58:01 (31). But no article has yet reported the relationship between B * 58:01 and AS. Further study is necessary to explore the association.
In the present study, we evaluated the HLA-B genotype in AS patients compared to the control group. As a result, we found that more than 98% of the samples were heterozygous in the HLA-B region. HLA-B27 homozygous patients were rare and only had axial manifestations. Based on our study and other reports, for B27(+) people, HLA-B40 can increase the risk of AS. HLA-B40 was the second most common HLA-B subtype in all of the AS patients besides HLA-B27. Then the genotype HLA-B27:04/B * 40:01 can improve diagnostic accuracy, and patients with HLA-B * 27:04/B * 46:01 had a high risk of arthritis and enthesitis. In the HLA-B27(−) group, HLA-B * 15:02 was a risk maker of peripheral joint involvement. Perhaps HLA-B * 40:01, HLA-B * 46:01, and HLA-B * 15:02 should be markers included in AS diagnosis value. Due to the limited information in this field and a small number of patients, our results did not show statistical significance in other HLA-B subtypes with peripheral clinical manifestations. There is a need for more samples and further workup on the relationship of the HLA-B heterozygous in AS patients.
In conclusion, our research shows that, besides HLA-B27, other HLA-B types also may impact the AS patient phenotype.
It is critical to systematically screen and evaluate the HLA-B genotype in the patients with AS, which may result in an improved accurate diagnosis of the patients.

DATA AVAILABILITY STATEMENT
The authors acknowledge that the data presented in this study must be deposited and made publicly available in an acceptable repository, prior to publication. Frontiers cannot accept a manuscript that does not adhere to our open data policies.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by The ethics committee of Third Affiliated Hospital of Sun Yat-Sen University. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
JG conceived the study and critically revised the manuscript and provided final approval of the manuscript. JW, PZ, and XL were in charge of the experiment. XW performed the analysis. XZ, ZC, QL, LT, QW, and SC were in charge of collecting sample and data. XW wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.