Cloning of the HLA class II region in yeast artificial chromosomes.

Yeast artificial chromosomes (YACs) have been applied to clone the entire class II region of the human major histocompatibility complex (MHC), including its flanking regions, in a contig over 1.5 million base pairs (bp) long. The human DNA inserts in the YACs have a size between 60 and 1300 kbp and were isolated from two EcoRI partial digest libraries. The gaps between DRA and DRB, DRB and DQA, and DOB and DPA, which had not been cloned by other means, have been bridged with YAC clones. The contig extends through the 400 kpb of DNA between the DRA and C4 genes, thus linking the class II region with the complement gene cluster in the class III region. The cloning in YACs has been supported by a conventional cosmid walk of 290 kbp in the C4-DRA region. Restriction enzyme sites in the YAC clones were compared to the sites in the cosmid walk, to published cosmid clones, and to the already existing physical maps, leading to a detailed characterization of a region of the human genome over 1500 kbp. The YAC clones will be valuable for functional analysis of the MHC.     

Measurement of HLA class I expression in ankylosing spondylitis.

The importance of HLA-B27 in the pathogenesis of ankylosing spondylitis is uncertain: current evidence favours a role for the B27 molecule itself. The possibility that quantitative differences in HLA-B27 expression may exist between patients with ankylosing spondylitis, family members, and control subjects positive for B27 was examined using appropriate monoclonal antibodies, flow cytometry, and a 'model lymphocyte' coated with a known number of mouse immunoglobulin binding sites. No differences were found between the groups. HLA-A2, examined for comparison, was expressed in greater amounts than HLA-B27, but each contributed only 10-20% of the total class I antigens. Homozygotes expressed twice the amount of antigen expressed by heterozygotes. Synovial lymphocytes expressed more class I antigens than peripheral lymphocytes.     

A gene in the HLA class I region contributes to susceptibility to IDDM in the Finnish population

Summary In Finland the haplotype A2, Cw1, B56, DR4, DQ8 is the third most common haplotype in insulin-dependent diabetic (IDDM) patients and has the highest haplotype-specific absolute risk for IDDM. Cw1, B56, DR4, DQ8 haplotypes containing HLA-A alleles other than A2 are infrequent in the population and are not associated with IDDM. Comparison of the A2 and non-A2 haplotypes at the DNA level showed that they were identical at HLA-B,-DR, and -DQ loci. Evidence that class I alleles confer susceptibility to IDDM was obtained from the two HLA-C, -B, -DR and -DQ haplotypes most frequently found in IDDM patients in Finland. A24, A3 and A2 on the Cw3, B62, DR4, DQ8 haplotype, and A28, A2 and A1 on the Cw7, B8, DR3, DQ2 were all found to be associated with IDDM. In Finland these seven haplotypes, including A2, Cw1, B56, DR4, DQ8, account for 33% of diabetic haplotypes and 10.3% of non-diabetic haplotypes (p<0.00001). The contribution of the class I region to IDDM susceptibility was also apparent in those IDDM patients lacking the disease-predisposing class II alleles. Significantly more non-DR3/non-DR4 IDDM patients (47 of 55) possessed two of the IDDM-associated HLA-A alleles compared to non-DR3/non-DR4 control subjects (40 of 58; p=0.038). Moreover, IDDM patients confirmed by oligotyping as unable to form a diabetes-susceptibility DQ heterodimer, tended to possess two diabetes-associated HLA-A alleles (12 of 13) compared to control subjects (12 of 20; p=0.056).Abbreviations IDDM Insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus - PCR polymerase chain reaction - SSO sequence specific oligonucleotide - TNF tumour necrosis factor     

HLA class I antibodies in thalassemic patients

This study was undertaken to demonstrate the prevalence of HLA class I antibodies among 62 polytransfused patients. The diagnosis included beta-thalassemia major, beta-thalassemia/Hb E disease and severe Hb H disease. Their ages ranged from 1 year to 23 years with the mean age of 10.7 years. The number of packed red cell transfusions ranged from 3 to 235 with the mean of 60 episodes per patient. The standard microlymphocytotoxicity test was performed using 50 panels of lymphocytes which specifically identified the majority of HLA class I antibodies. 31/62 cases (50%) were positive for HLA class I antibodies. The detection of single or multiple antibodies depended upon the number of blood transfusions and the patients' ages. These antibodies were induced by the leukocytes present in the transfused packed red cells. Therefore, leukocyte-reduced packed red cells prepared by either additional inverted centrifrugation or leukocyte filter is suggested for the routine blood bank service.     

A new marker in the HLA class I region is associated with the age at onset of IDDM

Summary The (MHC) class II association with insulin-dependent diabetes mellitus (IDDM) is well documented. However, it is likely that genes within the MHC class III and the class I region also play a role in determining susceptibility to IDDM. In this study we have used a novel molecular probe to investigate the class I P3A and P3B loci of 179 patients with IDDM and 142 normal control subjects. A highly significant increase in the frequency of the class I P3 4.0;1.5 kilobase (kb) and 4.0;1.8;1.5 kb genotypes was found in patients compared to the control subjects (x ² 46.8, 6 df, p<0.0001). The association with the P3B 1.5 kb allele was strongly associated with the age at onset of diabetes, being present in 96.2% of subjects who developed diabetes between the age of 10–20 years compared to 55.0 and 74.6% who developed diabetes before 10 years or after 20 years, respectively (x ² 31.4, p<0.0001). There was no evidence for linkage disequilibrium between the DQA1 and DQB1 loci and P3B suggesting that this is an independent association. In conclusion, these results suggest that genes in both the MHC class I and II regions confer susceptibility to IDDM and are related to the age at onset of the disease.Abbreviations kb Kilobase - SSC 0.3 mol/l sodium chloride, 0.3 mol/l sodium citrate - YAC yeast artificial chromosome - df degrees of freedom     

Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I-Cw4 or -Cw3 from untransfected or -Cw4 or -Cw3 alleles transfected HLA-A(-), B(-), C(-), G(-), E(+), F(+) 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR- or anti-KIR-specific monoclonal antibodies (mAbs). After sHLA-I-activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I-mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor-mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H(+)-adenosine triphosphatase (H(+)-ATPase) of granules, inhibited activating receptor-induced NK cell cytolysis, suggesting that activating receptor-mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-gamma (IFN-gamma) was detectable in culture supernatant of activating receptor(+) NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor-mediated IFN-gamma production. This suggests that activating receptor-induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-gamma, an antiviral cytokine able to amplify immune responses.     

Molecular dynamics of MHC genesis unraveled by sequence analysis of the 1,796,938-bp HLA class I region

The intensely studied MHC has become the paradigm for understanding the architectural evolution of vertebrate multigene families. The 4-Mb human MHC (also known as the HLA complex) encodes genes critically involved in the immune response, graft rejection, and disease susceptibility. Here we report the continuous 1,796,938-bp genomic sequence of the HLA class I region, linking genes between MICB and HLA-F. A total of 127 genes or potentially coding sequences were recognized within the analyzed sequence, establishing a high gene density of one per every 14.1 kb. The identification of 758 microsatellite provides tools for high-resolution mapping of HLA class I-associated disease genes. Most importantly, we establish that the repeated duplication and subsequent diversification of a minimal building block, MIC-HCGIX-3.8-1-P5-HCGIV-HLA class I-HCGII, engendered the present-day MHC. That the currently nonessential HLA-F and MICE genes have acted as progenitors to today's immune-competent HLA-ABC and MICA/B genes provides experimental evidence for evolution by "birth and death," which has general relevance to our understanding of the evolutionary forces driving vertebrate multigene families.     

HLA class I associations of ankylosing spondylitis in the white population in the United Kingdom.

OBJECTIVE: To investigate the HLA class I associations of ankylosing spondylitis (AS) in the white population, with particular reference to HLA-B27 subtypes. METHODS: HLA-B27 and -B60 typing was performed in 284 white patients with AS. Allele frequencies of HLA-B27 and HLA-B60 from 5926 white bone marrow donors were used for comparison. HLA-B27 subtyping was performed by single strand conformation polymorphism (SSCP) in all HLA-B27 positive AS patients, and 154 HLA-B27 positive ethnically matched blood donors. RESULTS: The strong association of HLA-B27 and AS was confirmed (odds ratio (OR) 171, 95% confidence interval (CI) 135 to 218; p < 10(-99)). The association of HLA-B60 with AS was confirmed in HLA-B27 positive cases (OR 3.6, 95% CI 2.1 to 6.3; p < 5 x 10(-5)), and a similar association was demonstrated in HLA-B27 negative AS (OR 3.5, 95% CI 1.1 to 11.4; p < 0.05). No significant difference was observed in the frequencies of HLA-B27 allelic subtypes in patients and controls (HLA-B*2702, three of 172 patients v five of 154 controls; HLA-B*2705, 169 of 172 patients v 147 of 154 controls; HLA-B*2708, none of 172 patients v two of 154 controls), and no novel HLA-B27 alleles were detected. CONCLUSION: HLA-B27 and -B60 are associated with susceptibility to AS, but differences in HLA-B27 subtype do not affect susceptibility to AS in this white population.     

Soluble HLA class I antigens in sera of patients with chronic hepatitis.

Soluble HLA Class I antigens in sera (serum-HLA Class I, s-HLA Class I) of patients with chronic hepatitis (CH) were measured with an enzyme-linked double determinant immunoassay (E-DDIA). The mean titers of s-HLA Class I antigens of patients with CPH (mean +/- standard deviation, 2.22 +/- 1.60), CAH2A (2.24 +/- 1.65) or CAH2B (2.73 +/- 1.46) were significantly higher than that of normal subjects (0.36 +/- 0.27) (P less than 0.01). The titer of s-HLA Class I correlated significantly with the level of serum glutamic pyruvic transaminase (s-GPT) (r = 0.73), and weakly with serum level of beta 2-microglobulin (r = 0.43). In patients with chronic hepatitis type B (CH-B) treated with human lymphoblastoid interferon alpha (IFN-alpha), the titer of s-HLA Class I antigens increased. The increased level of s-HLA Class I antigens in the clinical course of chronic hepatitis may be caused by their release from necrotizing hepatocytes which have acquired the expression of HLA Class I antigens on the cell-surface membrane during viral infection.     

HLA class III region and susceptibility to rheumatoid arthritis

OBJECTIVE: We examined the contribution of the HLA class III region in susceptibility to rheumatoid arthritis (RA). METHODS: Patients with RA, healthy subjects and homozygous typing cell (HTC) lines were typed for HLA class I (A, B, C), class II (DR, DQ) and class III (D6S273, Bat 2, and TNFa microsatellites, and HSP70 promoter region) alleles by molecular techniques. RESULTS: Based on the distribution of microsatellites D6S273, Bat2 and TNFa, and HSP70 promoter region alleles in HTCs and homozygous unrelated individuals, a class III region haplotype, D6S273 138-HSP70c-Bat2 138-TNFa2 was identified. This haplotype showed a significant primary association with susceptibility to RA in DRB 1 QKRAA/QRRAA epitope-negative patients. CONCLUSION: Since the QKRAA/QRRAA epitope does not provide any risk for disease susceptibility in RA-susceptibility DRB1 epitope-negative patients, the present data suggest that the class III region haplotype D6S273 138-HSP70c-Bat2 138-TNFa2 provides an additional risk for the development of RA. These results show that two regions in MHC, class II (DRB1) and class III (D6S273 138-HSP70c-Bat 2 138-TNFa2), contribute to susceptibility to RA and more completely define the risk for development of the disease.     

Platelet activation by cross-linking HLA class I molecules and Fc receptor.

The effect on platelet activation of monoclonal antibodies directed against common determinants of the HLA class I heavy chain molecule was studied. Cross-linking W6/32, an anti-HLA class I of IgG2a subclass, led to platelet activation. Two other antibodies of the same subclass did not have this effect on platelets. The lack of activity of the F(ab')2 fragments suggests that the activation signal is mediated by the platelet Fc receptor (Fc gamma RII). Indeed, except for a higher sensitivity of W6/32 to aspirin and apyrase, activations by cross-linking IV-3 (an anti-Fc gamma RII) and W6/32 are similar at the level of InsP3 formation, calcium mobilization, pH modifications, and activation of protein kinase C and myosin kinase. When HLA class I molecules and Fc gamma RII are cross-linked together, platelet activation occurs. This is not observed when a control IgG2a is substituted for W6/32 or when CD9 and Fc receptor are cross-linked together. This suggests that HLA class I molecules and Fc gamma RII synergize to activate platelets.     

Molecular typing of HLA class I and class II antigens in Indian kala-azar patients

HLA has been shown to be associated with many diseases. To find out whether host genetic factors like the HLA are involved in susceptibility to kala‐azar (visceral leishmaniasis) in India, we formulated an association study with genetically related controls. All samples were typed by PCR SSOP (sequence specific oligonucleotide probes) for HLA class I (A and B) and class II (DR) antigens. The test of association we used was the transmission disequilibrium test (TDT). No significant evidence for association with any of the three HLA loci was obtained.     

Clinical relevance of soluble HLA class I molecules in Waldenstrom Macroglobulinemia

Objectives: Waldenstrom Macroglobulinemia (WM) is a B‐cell neoplasm characterised by secretion of IgM by lymphoplasmacytic bone marrow cells and by cytopenias and hypogammaglobulinemia in a subset of patients. Beta‐2 microglobulin (b2m) is a major prognostic factor in WM and the heavy chain of HLA class I molecules, which are known to have immunosuppressive properties and have been implicated in the pathogeny of several malignancies. Methods: We assessed the serum levels of the total soluble HLA‐I molecules and the HLA‐Gs molecules in 105 patients with IgM‐related disorders [WM (n = 42) and IgM MGUS (n = 63)], and compared the results to 41 healthy subjects. Results: We found higher levels of HLA‐Is in WM, compared to IgM MGUS and healthy donors. HLA‐Gs levels were similar in WM and in IgM MGUS, but higher than in healthy donors. The association between HLA‐Is at the cut‐off of 1.8 μg/mL and known markers of poor prognosis was then evaluated among WM patients using univariate and multivariate methods. Based on this, high HLA‐Is level was strongly associated with high serum β2M level >3 mg/L [OR = 2, (CI 95% 1.1–5.7); P = 0.04], age > 65 yrs [OR = 1.5, (CI 95% 0.5–4.1), P = 0.06] and haemoglobin ≤11.5 g/dL [OR = 3.3, (CI 95% 1.2–9.7); P = 0.03]. High levels of serum HLA‐Is were also found in patients with cryoglobulinemia, however irrespectively of WM or IgM‐MGUS status. Conclusion: Together our results suggest a possible role for soluble MHC class I molecules in WM disease. Further investigations are necessary to further demonstrate the prognostic impact of soluble MHC class I molecules in Waldenstrom Macroglobulinemia.     

The role of HLA class I gene variation in autoimmune diabetes

The use of DNA-based genetic typing has enabled the identification of type 1 diabetes mellitus (T1DM) susceptible and protective major histocompatibility complex (MHC) class II alleles and haplotypes. The application of this approach has also progressed to locate MHC class I alleles that contribute to the clinicopathology of T1DM. Recent studies have shown a widespread involvement of genes from the MHC class I gene region in the clinicopathology of T1DM. These genes are shown to be involved in contributing to progression from the preclinical stage of the disease, which is characterized by the occurrence of islet-specific antibodies, to clinical disease and also to the occurrence of autoimmunity. They can either contribute directly to disease development or indirectly in concert with other susceptible MHC class II alleles or haplotypes via linkage disequilibrium. Class I alleles may also be negatively associated with T1DM. These findings are useful for the development of future strategies in designing tolerogenic approaches for the prevention or even reversal of T1DM. In this article, the latest evidence for the different kinds of participation of HLA class I genes in the etiology of T1DM is reviewed. A meta-analysis which included existing association studies was also carried out in order to re-assess the relevance of class I genes in diabetes development. The analysis of an enlarged heterogeneous sample confirmed the involvement of previously detected serotypes in the etiology of T1DM, such as A24, B8 and B18, and revealed hitherto unknown associations with B60 and B62. The analysis points out that much of the conflicting results of previous association studies originate from inadequate sample sizes and accentuate the value of future investigations of larger samples for identifying linkage in multigenic diseases.     

HLA class I and class II antigens associated with multiple myeloma in southern Africa

While the exact aetiology of myeloma is unknown, genetic factors feature among the potential risk factors. The HLA phenotypes in African blacks with myeloma (the commonest haematopoietic malignancy in this group) have not been characterized. The purpose of this study was to determine the HLA class I and class II phenotypes of patients with multiple myeloma and to compare the findings to an ethnically matched control group of 100 individuals. Analysis of the HLA class I and class II phenotypes in 62 myeloma patients revealed: (i) a corresponding statistically significant association with HLA B18 [odds ratio (OR) 6.3; 95% confidence interval (CI) 1.013-39.727; P < 0.005]; (ii) no statistically significant association with HLA B13, Cw2, Cw6 or the DR and DQ antigens; and (iii) a statistically significant negative (protective) association with HLA Cw7 (OR 0.4; 95% CI 0.21-0.87; P < 0.005). This study suggests that although genetic factors may play a role in the multifactorial aetiology of multiple myeloma, with the exception of HLA B18, there is no specific association between HLA types and multiple myeloma in South African blacks.