High-resolution HLA-A, -B and -DRB1 allele and haplotype frequencies in 7823 Han marrow donors of Liaoning province, China.

BACKGROUND: The human leukocyte antigen (HLA) system is the most polymorphic gene cluster in humans. High-resolution donor-recipient matching for HLA genes improves patient survival after unrelated hematopoietic stem cell transplantation. MATERIALS AND METHODS: In this study, we analyzed the high-resolution allele and haplotype frequencies at the HLA-A, -B and -DRB1 loci in the Liaoning Han population and analyzed its relationships with other populations. RESULTS: The 3 most frequent alleles at the HLA-A, -B and -DRB1 loci were A*24:02, A*02:01:01G, A*11:01; B*13:02, B*46:01, B*40:01:01G; DRB1*09:01, DRB1*15:01 and DRB1*07:01, respectively. The most frequent 2-locus haplotypes were A*30:01-B*13:02 and B*13:02-DRB1*07:01. A*30:01-B*13:02-DRB1*07:01 was determined to be the predominant 3-locus haplotype. Hot maps and multiple correspondence analyses based on the frequencies of HLA specificities, which allow statistical visualization of dependent and independent relationships among variables, indicate that the Liaoning Han population is closely related to Northern populations of China and shows relative close relationships with Asian populations. CONCLUSION: These data will provide an outline of the HLA characteristics of healthy individuals in our region and help bone marrow transplantation patients find suitable HLA-matched donors.

Polymorphisms in the F Pocket of HLA-B27 Subtypes Strongly Affect Assembly, Chaperone Interactions, and Heavy-Chain Misfolding.

OBJECTIVE: HLA-B27 is associated with the inflammatory spondyloarthritides (SpA), although subtypes HLA-B*27:06 and HLA-B*27:09 are not. These subtypes differ from the HLA-B*27:05 disease-associated allele primarily at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen-binding groove. Dimerization of HLA-B27 during assembly has been implicated in disease onset. The purpose of this study was to investigate the factors that influence differences in dimerization between disease-associated and non-disease-associated HLA-B27 alleles. METHODS: HLA-B*27:05 and mutants resembling the HLA-B*27:06 and 09 subtypes were expressed in the rat C58 T cell line, the human CEM T cell line and its calnexin-deficient variant CEM.NKR. Immunoprecipitation, pulse-chase experiments, flow cytometry, and immunoblotting were performed to study the assembly kinetics, heavy-chain dimerization, and chaperone associations. RESULTS: By expressing HLA-B*27:05, 06-like, and 09 alleles on a restrictive rat transporter associated with antigen processing background, we demonstrate that a tyrosine expressed at p116, either alone or together with an aspartic acid residue at p114, inhibited HLA-B27 dimerization and increased the assembly rate. F-pocket residues altered the associations with chaperones of the early major histocompatibility complex class I folding pathway. Calnexin was demonstrated to participate in endoplasmic reticulum (ER) stress-mediated degradation of dimers, whereas the oxidoreductase ERp57 does not appear to influence dimerization. CONCLUSION: Residues within the F pocket of the peptide-binding groove, which differ between disease-associated and non-disease-associated HLA-B27 subtypes, can influence the assembly process and heavy-chain dimerization, events which have been linked to the initiation of disease pathogenesis.

Determination of IL-23 receptor gene polymorphism in Iranian patients with ankylosing spondylitis.

INTRODUCTION: The result of recent genome-wide association studies revealed that, in addition to HLA-B27, a few non-HLA genes are associated with susceptibility to ankylosing spondylitis (AS) in Caucasian populations. According to these studies, IL-23R is one of the genes that is associated with AS. In this study, we evaluated five important single nucleotide polymorphisms (SNPs) of the IL-23R gene which confers susceptibility to AS, and its effects on the severity of the disease in HLA-B27 positive and negative patients and several subtypes of HLA-B27. MATERIALS AND METHODS: The study population consisted of 294 AS patients and 352 age-, sex-, and ethnicity-matched healthy controls. All patients were examined by rheumatologists, and met modified, New York criteria for the disease. Five SNPs (rs1004819, rs11209032, rs1495965, rs11465804, and rs1004819) of the IL-23R gene were genotyped using the Real-Time PCR TaqMan genotyping method. RESULTS: We found that only rs1004819 has a significant association with AS, and that the remaining four SNP alleles are not associated with AS. Also, there was no association between these five polymorphisms and BASDAI, BASFI, and BASMI indices. Two haplotypes, ACGAT and ACGAG, were found to be associated with the heritability of AS. In addition, two significant, protective diplotypes (D8, GCGAG/GTGGG ; and D9, ACGAG/GCGAG) were discovered. CONCLUSION: This study supported our previous findings regarding the differences between the genetic patterns of AS in Iranian patients compared with those in other parts of the world.

HLA-B*27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T cell epitope.

BACKGROUND & AIMS: HLA-B*27 is associated with spontaneous HCV genotype 1 clearance. HLA-B*27-restricted CD8+ T cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B*27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B*27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. METHODS: Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B*27+ patients with chronic HCV genotype 1 infection. CD8+ T cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B*27:02 and 05. RESULTS: The NS5B2820 epitope is only restricted by the HLA-B*27 subtype HLA-B*27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B*27 subtype B*27:05. Indeed, the epitope is very dominant in HLA-B*27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B*27:02+ chronically infected patients. CONCLUSIONS: The NS5B2820 epitope is immunodominant in the context of HLA-B*27:02, but is not restricted by other HLA-B*27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines.

Effect of HLA-B*27 and its subtypes on clinical manifestations and severity of ankylosing spondylitis in Iranian patients.

The aim of this study was to assess the role of HLA-B*27 and it's subtypes in determining severity and clinical manifestations of ankylosing spondylitis (AS).A total of 163 AS patients were assessed for clinical manifestations and severity using structured questionnaires. HLA-B*27 screening and B*27 sub-typing were performed by PCR.One hundred twenty two patients (74.8%) were B*27 positive. The male to female ratio, peripheral arthritis, steroid use, intense dorsal kyphosis and decrease of cervical slope had a significantly higher frequency in B*27 positive patients compared to B*27 negative ones (p=0.01, 0.001, 0.01, 0.04 and 0.04, respectively). However, the age of diagnosis was significantly lower in B*27 positive patients (p=0.005). Trend in uveitis and some severity markers including: BASMI and ASQoL were toward higher values in B*27 positive group with no significant difference. After controlling confounding variables, significant relationship was found only between B*27 and BASMI (p=0.01). B*27 subtypes in patients were included B*2705: 48.4%, B*2702: 42.6%, B*2704: 5.7% and B*2707: 3.3%. No significant differences were seen for severity markers and clinical manifestations between subtypes; although trend toward lower values of severity markers, less intense dorsal kyphosis and less decrease of cervical slope were observed in B*2704 and B*2707 versus other polymorphisms.Clinical features and severity of AS is influenced by HLA-B*27. Trend toward higher severity markers in B*2705 and B*2702 versus other polymorphisms might be subject of interest for evaluation in other ethnicities with concentration to other novel susceptibility genes co-inherited in each B*27 subtype.

Structural and dynamic features of HLA-B27 subtypes.

PURPOSE OF REVIEW: The differential association of HLA-B27 subtypes with ankylosing spondylitis provides the rationale for a comparative investigation of these proteins. Results from the last 2 years of research on minimally distinct HLA-B27 subtypes, primarily using biochemical and biophysical techniques, are presented and discussed. RECENT FINDINGS: We summarize evidence that micropolymorphisms within the molecules' peptide-binding groove influence wide-ranging biochemical, biophysical and antigenic properties of HLA-B27 molecules, and suggest that distinct, subtype and peptide-dependent dynamics of peptide - heavy chain - ß(2)-microglobulin heterotrimers could be instrumental for an understanding of the initiation of disease processes that are connected with certain HLA-B27 subtypes. SUMMARY: The results indicate that mAbs that bind only to structurally distinguishable subsets of HLA-B27 molecules as well as techniques that assess the flexibility of these antigens may hold the key to comprehend molecular events contributing to the initial stages of disease pathogenesis in spondyloarthropathies.

HLA B27 subtype distribution among patients with ankylosing spondylitis in eastern Turkey.

Human leukocyte antigen (HLA) B27 has a strong association with ankylosing spondylitis (AS) and other spondyloarthropathies. More than 70 subtypes of HLA B27 have been described. The present study investigated B27 subtype distribution among B27-positive patients with AS classified according to the modified New York criteria and healthy controls. Sequence-specific primer polymerase chain reaction technique was used for B27 subtyping of 43 unrelated patients with AS and 39 volunteer bone marrow donors. Among patients with AS, the male-female ratio was 6.2 and the mean age was 30 years. No relationship was found between the B27 subtypes and clinical and laboratory findings in patients with AS (p>0.05). Similarly, the frequencies of B27 subtypes did not significantly differ between patients and controls. In this study, B*2746, B*2749, and B*2767 subtypes were detected for the first time. Among B27 subtypes, the most common B27 alleles found in the patients and the controls were B*2702 and B*2705. In addition, B*2702 subtype was found predominantly in both patients (48.8%) and controls (46.2%). In conclusion, in addition to commonly encountered B*2702 and B*2705 HLA subtypes, a B*2749 subtype in a patient with AS and B*2746 as well as B*2767 subtypes in controls were determined for the first time.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) exhibits functionally significant interaction with HLA-B27 and relates to subtype specificity in ankylosing spondylitis.

OBJECTIVES: The functional interaction of endoplasmic reticulum aminopeptidase 1 (ERAP1) with human leucocyte antigen (HLA)-B*27 could be important in the pathogenesis of ankylosing spondylitis (AS). AS is associated with B*27:04 and B*27:05, but not with B*27:06 and B*27:09. The authors studied the surface expression of peptide-HLA(pHLA)-B27 complexes and HLA class-I free heavy chains (FHCs) on peripheral blood mononuclear cells of patients with AS with different ERAP1 single nucleotide polymorphisms. The effects of ERAP1 suppression on HLA-B*27 subtypes were tested. METHODS: Peripheral blood mononuclear cells were collected from Caucasian patients with AS for flow cytometry and were stained for pHLA and FHCs. Genotyping was performed for two ERAP1 single nucleotide polymorphisms (rs27044(C/G) and rs30187(C/T)). C1R cells transfected with different HLA-B27 subtypes (B*27:04, B*27:05, B*27:06 and B*27:09) were subjected to ERAP1 suppression by small interfering RNA and stained using the monoclonal antibody (mAb) MARB4 as well as antibodies for pHLA, FHC, intracellular FHC (IC-FHC). MARB4 has been reported to bind to HLA-B27 with extended peptides. RESULTS: The authors found variations in FHC expression on the monocytes of patients with AS, depending on different ERAP1 variants. Subsequently, using Hmy2.C1R cells in vitro, the authors show that ERAP1 suppression leads to increased IC-FHC and surface pHLA that react with the monoclonal antibody MARB4. The functional interaction between ERAP1 and HLA-B27 molecules appears to be subtype-specific, since ERAP1 suppression leads to changes only in cells expressing B*27:04 or B*27:05, but not B*27:06 or B*27:09. CONCLUSIONS: Direct or indirect alterations in the ERAP1-HLA-B27 interaction could be crucial by causing changes in peptide presentation or FHC formation by HLA-B27 molecules, as well as by contributing to differential subtype association in spondyloarthropathies.

The prevalence of HLA-B27 in the US: data from the US National Health and Nutrition Examination Survey, 2009.

OBJECTIVE: To carry out the first large-scale population study of the prevalence of HLA-B27 in the US, which is needed for public health planning purposes because of recent improvements in medical therapy and diagnostic testing for ankylosing spondylitis (AS). METHODS: The national prevalence of HLA-B27 was determined as part of the 2009 US National Health and Nutrition Examination Survey (NHANES), a cross-sectional survey monitoring the health and nutritional status of the US civilian, noninstitutionalized population. DNA polymerase chain reaction analysis was conducted in samples from 2,320 adults ages 20-69 years from this nationally representative sample. RESULTS: The age-adjusted US prevalence of B27 was 6.1% (95% confidence interval [95% CI] 4.6-8.2). By race/ethnicity, the prevalence of B27 was 7.5% (95% CI 5.3-10.4) among non-Hispanic whites and 3.5% (95% CI 2.5-4.8) among all other US races/ethnicities combined. In Mexican Americans, the prevalence was 4.6% (95% CI 3.4-6.1). The prevalence of B27 could not be reliably estimated for other US racial/ethnic groups because of the low number of B27-positive individuals in those groups. For adults 50-69 years of age, the prevalence of B27 was 3.6% (95% CI 2.2-5.8), which suggested a decrease in B27 with age. These prevalence estimates took into account the NHANES survey design and are reviewed with respect to data from the medical literature. CONCLUSION: Our findings provide the first US national prevalence estimates for HLA-B27. A decline in the prevalence of HLA-B27 with age is suggested by these data but must be confirmed by additional studies.

A single-nucleotide polymorphism marker within the FCRL5 gene and HLA-B27 positive Han Chinese ankylosing spondylitis patients.

The aim of this study was to determine whether FCRL5 genes in concert with human leukocyte antigen-B27 (HLA-B27) genotypes are associated with susceptibility to ankylosing spondylitis (AS) in Chinese population. One hundred and sixty-nine HLA-B27-positive AS patients (107 males and 62 females) and 184 HLA-B27-positive matched controls (112 males and 72 females) were analyzed from Han Chinese populations by case-control design, and their samples were genotyped using a panel of two single-nucleotide polymorphism (SNP) markers (rs6427384, rs12036228) within the FCRL5 gene by ligase detection reactions (LDRs) and the HLA-B27 subtypes were determined by polymerase chain reaction (PCR) using sequence-specific primer (SSP) methods. Our results show that in addition to B27, the SNPs rs6427384 and rs12036228 were associated with AS, and the C-T haplotype was higher in cases with AS than in the control population [74.8% vs 63.6%, Fisher's P = 0.003, odds ratio (OR) = 1.660,95% confidence interval (CI) = 1.184-- 2.326]. Our results suggest that, in addition to HLA-B27, a novel polymorphism within the FCRL5 gene confers susceptibility to AS in Han Chinese population.

HLA-B27 subtypes and tumor necrosis factor alpha promoter region polymorphism in Iranian patients with ankylosing spondylitis.

BACKGROUND: HLA-B27 is an MHC class I molecule that is strongly associated with ankylosing spondylitis (AS). TNF-alpha, as an important cytokine in inflammatory joint disease, might have a role in the process of AS. This study was performed to determine HLA-B27 subtypes among Iranian patients with AS, and to investigate TNF-alpha gene polymorphisms in the patient groups. METHODS: Ninety seven AS patients (74 HLA-B27-positive and 23 HLA-B27-negative) and 137 healthy normal subjects (2 HLA-B27-positive) were enrolled in this study. HLA-B27 positive patients were screened using the polymerase chain reaction, with sequence specific primers (PCR-SSP), for B*27 subtyping. All patients and the controls were also investigated for determination of TNF-alpha polymorphisms using the same method. RESULTS: Just two subtypes were detected in our patients, namely B*2705 (63.4%) and B*2702 (36.6%). The study of TNF-alpha polymorphisms at position -238 showed that the A allele and AA genotype were significantly over-represented in all patient groups in comparison with the control group (p < 0.001). At position -308, while the A allele and AA genotype were significantly over-represented in the whole patient group (p = 0.01), there was no significant difference between the AS groups and the control group. CONCLUSION: It could be suggested that a polymorphism within the TNF-alpha gene at -238 play an important role in AS, although this polymorphism was not related to HLA-B27 subtypes.

Application of the particle gel agglutination assay in the typing of single human leucocyte antigens.

We describe a simple and rapid particle gel agglutination assay (PaGIA) for typing of the human leucocyte antigens (HLA) HLA-A2, HLA-B7 and HLA-B27. Superparamagnetic streptavidin particles were coated with biotinylated monoclonal antibodies (MoAbs) to HLA-A2, HLA-B7 and HLA-B27. Anticoagulated whole blood samples from healthy blood donors (n = 118) with known HLA patterns were incubated with MoAb-coated particles, transferred into a standard ID-gel card, and subsequently centrifuged. Samples were evaluated macroscopically, with antigen-positive samples resulting in a visible agglutination reaction. A clear distinction could be made between all positive and negative samples tested. Fifty-seven samples were found to be positive for HLA-A2 (48%), 26 samples for HLA-B7 (22%) and 5 samples for HLA-B27 (4%).

[Prokaryotic expression of HLA-B * 2705 heavy chain and identification for its activity].

In order to investigate the expression of heavy chain of HLA-B * 2705 in prokaryotic system and identify its activity, the extra-membrane gene fragment of HLA-B * 2705 was amplified from full-length HLA-B*2705 cDNA by PCR and cloned into pGEM-T vector. After identification by sequencing, the prokaryotic expressing vector pET32a (+)-B * 2705 was constructed. The antigenic activity of expressed protein was identified by Western blot and antibody blocking reaction. The results indicated that the fused HLA-B * 2705 protein expression with high efficiency was obtained. The expressed product was more than 50% of the total bacteria protein. The antigenic activity of expressed protein was confirmed by Western blot and antibody blocking reaction. It is concluded that HLA-B * 2705 fusion protein are obtained as basis for the further studies.

Usefulness of human leucocyte antigen-B27 subtypes in predicting ankylosing spondylitis: Taiwan experience.

BACKGROUND: Genetic factors are clearly attributed to the susceptibility of ankylosing spondylitis (AS). The human leucocyte antigen (HLA)-B27 proved to be the very useful marker for diagnosing AS. The aim of this study was to determine the prevalence of HLA-B27 subtypes in Taiwan and to investigate whether these subtypes may be of help in predicting the diagnosis of AS. METHODS: A total of 314 patients with AS and a control group of 71 subjects positive for HLA-B27 detected by flow cytometry analysis were recruited for the study. HLA-B27 subtypes were confirmed by the polymerase chain reaction-sequence-specific primers and sequence-specific oligonucleotide probing. RESULTS: Four B27 alleles were identified: B*2704, B*2705, B*2706 and B*2707. HLA-B*2704 was the predominant allele. There were significant differences in the distribution of HLA-B27 subtypes between patients with AS and controls. Five of them who were homozygous for the B*2704 allele were solely found in AS group but not in controls. Statistical analysis showed that B*2704 was positively associated with AS, which suggested an increased possibility of having AS. Other HLA-B27 subtypes showed no strong correlation with AS. CONCLUSION: In the Taiwanese population, susceptibility to AS was determined by the presence of HLA-B*2704. Although B*2706 was reported to have a negative association with AS in Taiwanese, Thai and Chinese Singaporean populations, we report, in our study, two AS patients with B*2706 (0.6%). Disease heterogeneity suggests that other than genetic background, many pathogenic factors could be associated with AS. This may need to be investigated with a larger group of patients with AS and controls.

The pathogenetic role of HLA-B27 and its subtypes.

The strong association between HLA-B27 and ankylosing spondylitis has been known for more than 33 years, but the enigma of the pathogenetic role of the gene and its product has not yet been solved. Ongoing studies have produced evidence supporting different theories to explain this association, and structural and functional studies of HLA-B27 allele products at molecular level have provided information of broad and multidisciplinary value and disclosed new avenues leading to autoimmunity and immune disregulation.

[Frequencies distribution of human leukocyte antigen-B27 subtypes in healthy Chinese].

OBJECTIVE: To investigate the frequencies distribution of human leukocyte antigen (HLA)-B27 subtypes in unrelated healthy Chinese. METHOD: Polymerase chain reaction sequence-based typing (PCRSBT) was used to determine HLA high-resolution genotypes of 825 unrelated healthy Chinese. RESULTS: A total of 25 HLA-B27-positive individuals and 8 HLA-B27 subtypes were detected. These subtypes and their corresponding frequencies were B * 2704 (30.77%) , B * 2705 (23.08%), B * 2707 (19.23%), B * 2711 (7.69%), B * 2712 (7.69%), B * 2701 (3.85%), B * 2713 (3.85%) and B * 2721 (3.85%). CONCLUSION: The data obtained through PCR-SBT method may serve as important reference for the research of relationship between HLA-B27 subtypes and some diseases such as ankylosing spondylitis.

HLA-B27 typing: evaluation of an allele-specific PCR melting assay and two flow cytometric antigen assays.

BACKGROUND: Human leukocyte antigen B27 (HLA-B27) is a major histocompatibility complex class 1 molecule that is strongly associated with the disease ankylosing spondylitis. Testing for HLA-B27 is of diagnostic value because 90% of patients with ankylosing spondylitis have the B27 antigen. Two commonly used HLA-B27 flow cytometric assays are commercially available. METHODS: An allele-specific polymerase chain reaction (PCR) melting assay for HLA-B27 was compared with two available antigen assays on 371 clinical samples. The accuracy of the assays was measured by receiver operating characteristic analysis using the PCR method and sequencing as the reference standard. RESULTS: When PCR results were compared with those of the antigen assays, complete concordance was observed except for five discrepant results that were resolved by sequence analysis. Using DNA sequencing as the gold standard, the sensitivity and specificity of PCR were 99.6 and 100.0, those of the best single antigen assay were 98.2 and 97.6, and those of a reflex combination of both antigen assays were 98.8 and 97.6. CONCLUSIONS: The allele-specific PCR melting assay for HLA-B27 genotyping is easy to perform and has better sensitivity and specificity than antigen assays. The performance of the two flow cytometric antigen assays depends on the antibody used and the positive cutoff values assigned.

HLA-B27 in the Greek Cypriot population: distribution of subtypes in patients with ankylosing spondylitis and other HLA-B27-related diseases. The possible protective role of B*2707.

The major purpose of the present study was to investigate the frequency of human leukocyte antigen (HLA)-B27 alleles in healthy controls and in patients with ankylosing spondylitis (AS) and other HLA-B27-related diseases in the Greek Cypriot population. We selected 102 HLA-B27-positive individuals (60 controls and 42 patients). Typing of the HLA-B27 alleles was performed by polymerase chain reaction amplification with sequence-specific primers. Only two alleles were detected in the patient group: B*2702 (n = 31, 73.8%) and B*2705 (n = 11, 26.2%). The HLA-B*2707 allele was detected (n = 10, 16.7%) only in the healthy controls in addition to the B*2702 (n = 31, 51.7%) and B*2705 (n = 19, 31.7%) alleles. Our results show a restricted number of HLA-B27 subtypes associated with AS and other B27-related diseases and an elevated frequency of the B*2702 allele in the AS patients. The allele B*2707 seems to have a protective role in the population studied because it was found only in the healthy controls.