Resolution of HLA-B*44:02:01G, -DRB1*14:01:01G and -DQB1*03:01:01G reveals a high allelic variability among 12 European populations.

Within the framework of the EU-funded HLA-NET action, an analysis of three G-group alleles, HLA-B*44:02:01G, DRB1*14:01:01G and DQB1*03:01:01G, was undertaken in 12 European populations. Ambiguities were resolved by polymerase chain reaction-sequence-specific amplification (PCR-SSP) or PCR-sequence-based typing (PCR-SBT) in a total of 5095 individuals. The results of the DRB1*14:01/14:54 ambiguity showed high relative ratios (24-53%) of DRB1*14:01 in Bulgarians, Croatians, Greeks, Italians and Slovenians, contrasting with low ratios (6-13%) in Austrians, Finnish, French, Hungarians, Norwegians and Swiss. Resolution of the B*44:02/44:27 ambiguity showed that B*44:27 had a high relative ratio in Slovenians (25.5%) and Bulgarians (37%) and low in French and Swiss (0.02-1%), and was not observed in Greeks and Italians. The highest relative ratio of DQB1*03:19 was found in Portuguese (11%), by contrast with low ratios (0-3%) in the other five populations. Analysis of the A, B, DRB1 phenotypes and family-derived haplotypes in 1719 and 403 individuals positive for either HLA-B*44:02G or DRB1*14:01G ambiguities, respectively, showed some preferential associations, such as A*26∼DRB1*14:01, B*35∼DRB1*14:01, B*38∼DRB1*14:01 and B*44:27∼DRB1*16. Because these ambiguities are located outside the peptide-binding site, they may not be recognized by alloreactive T-cells. However, because of strong linkage disequilibrium (LD), the DRB1*14:01 vs DRB1*14:54 and the B*44:02 vs B*44:27 mismatches are associated to DRB3-, and C-mismatches, respectively. These results are informative for algorithms searching unrelated hematopoietic stem cell donors. For B*44:27-positive patients, searches are expected to be more successful when requesting donors from Southeastern-European ancestry. Furthermore, the introduction of human leukocyte antigen (HLA)-typing strategies that allow resolving exon 4 (for class I) and exon 3 (for class II) polymorphisms can be expected to contribute significantly to population genetics studies.

The role of novel agents on the reversibility of renal impairment in newly diagnosed symptomatic patients with multiple myeloma.

The role of thalidomide, bortezomib and lenalidomide in multiple myeloma patients presenting with renal impairment was evaluated in 133 consecutive newly diagnosed patients who were treated with a novel agent-based regimen. A significant improvement of renal function (renalPR (renal partial response)) was observed in 77% of patients treated with bortezomib, in 55% with thalidomide and in 43% with lenalidomide (P=0.011). In multivariate analysis, bortezomib-based therapy was independently associated with a higher probability of renal response compared with thalidomide- or lenalidomide-based therapy. Other important variables included eGFR (estimated glomerular filtration rate) ≥30 ml/min, age ≤65 years and myeloma response. Patients treated with bortezomib achieved at least renalPR in a median of 1.34 months vs 2.7 months for thalidomide and >6 months for lenalidomide-treated patients (P=0.028). In multivariate analysis bortezomib-based therapy, higher doses of dexamethasone (≥160 mg during the first month of treatment), an eGFR ≥30 ml/min and age ≤65 years were independently associated with shorter time to renal response. In conclusion, bortezomib-based therapies may be more appropriate for the initial management of patients with myeloma-related renal failure; however, thalidomide and lenalidomide are also associated with significant probability of improvement of their renal function.

Extensive bone marrow infiltration and abnormal free light chain ratio identifies patients with asymptomatic myeloma at high risk for progression to symptomatic disease.

Asymptomatic multiple myeloma (AMM) is characterized by a constant risk of progression to symptomatic myeloma. To evaluate previously recognized risk factors and to identify high-risk features we analyzed 96 patients with AMM and at least 18 months of follow-up. The progression rate at 1,2, and 3 years was 8%, 15% and 26%, respectively, and the projected 5-year progression rate was 38%. Extensive bone marrow (BM) infiltration, abnormal free light chain (FLC) ratio and serum monoclonal (M)-protein ≥ 3 gr/dl were the most significant factors for progression, whereas the type of heavy (IgG vs IgA) or light chain or immunoparesis of the uninvolved immunoglobulins were not. Abnormal marrow signal of magnetic resonance imaging of the spine was associated with a significant risk of progression (median 15 months, P=0.001). Extensive BM infiltration ≥ 60% (hazard ratio, HR: 13.7, P<0.001) and FLC ratio ≥ 100 (HR: 9, P=0.003) independently identified a 'very high-risk' group, which included 12.5% of patients with AMM and who progressed ≤ 18 months from initial diagnosis. Development of anemia and/or lytic bone lesions were the most common features of symptomatic progression. In conclusion, there is a subgroup of patients who have a substantial risk of progression to symptomatic disease that can be detected at diagnosis (either by extensive BM infiltration ≥ 60% or FLC ratio ≥ 100) and may be considered for immediate treatment.

HLA-DQA1*0505 sharing and killer immunoglobulin-like receptors in sub fertile couples: report from the 15th International Histocompatibility Workshop.

This aim of the study was to investigate whether human leukocyte antigen (HLA)-DQA1*0505 sharing or the maternal killer immunoglobulin-like receptor (KIR) repertoire is associated with recurrent spontaneous abortion (RSA) or repeated implantation failure (RIF). The study included 224 couples with RSA, 61 couples with RIF, 182 fertile couples, and 10 couples with successful in vitro fertilization and embryo transfer (IVF)/ET at first cycle. HLA-DQA1*0505 typing using polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) was performed in 185 RSA (117 with alloimmune abnormalities and 68 of autoimmune etiology), 61 RIF and 182 control couples, and KIR genotyping using polymerase chain reaction-sequence-specific primer (PCR-SSP) in 167 RSA and 55 RIF cases as well as 46 RSA and 10 IVF controls. No differences in DQA1*0505 sharing were found between patients and controls. In RSA and RIF women, the ratio of inhibitory to activating KIRs was slightly lower (1.53 and 1.85 vs 2.03 in controls). The analysis of maternal inhKIR and fetal HLA-C molecule pairs showed that the 'less inhibiting' combination KIR2DL3-C1 was found in higher percentage in subfertile (mainly RIF) than in fertile couples. In contrast, the percentage of cases possessing the 'strong inhibiting' combination KIR2DL1-C2 was lower in the RSA and RIF groups in comparison with that in the control groups (17.36% vs 23.91 and 16.36% vs 40%, respectively). In women with >or= 6 implantation failures, the KIR2DL1-C2 combination was not found in any of them (P = 0.0014), and the KIR2DL3-C1 combination was not found in the control IVF group. The results oppose the suggestion that increased HLA-DQA1*0505 sharing predispose to RSA or RIF. The KIR2DL3-C1 combination (or lack of the KIR2DL1-C2 one) is associated with implantation failure.

Takayasu arteritis: epidemiological, clinical, and immunogenetic features in Greece.

OBJECTIVE: Takayasu arteritis (TA) is an uncommon disease with clinical heterogeneity across different ethnic groups. We aimed to evaluate the epidemiological, clinical, and immuno-genetic features of TA in Greece. METHODS: Demographic, clinical, laboratory, angiographic, and therapeutic data of 42 patients from 4 large referral centers were retrieved. Serology and Human Lymphocyte Antigen (HLA) typing was performed in 22 patients. RESULTS: We studied 37 women and 5 men with a median age of 31 years at disease onset. Median delay in diagnosis was 24 months and median follow-up was 47 months (range 0-178). Constitutional or musculoskeletal symptoms were present in 86%, especially early in the disease course. Vascular findings were universal with reduced or absent pulse being the most common manifestation (98%). Hypertension was frequent (78%). Extensive disease prevailed and stenotic lesions were more common than aneurysms (95% vs. 40%). Erythrocyte sedimentation rate and C-reactive protein showed modest correlation with disease activity. HLA-B52 was expressed by 37% of the patients vs. 2.4% of the controls (p<0.001). Glucocorticoids and cytotoxic agents were used in most patients with remission rates of 83%. A total of 42 surgical procedures were performed with success rates of 87%. CONCLUSION: TA in Greece clinically and epidemiologically resembles the pattern of disease in Japan and the Western hemisphere. There is considerable delay in diagnosis, which may partially reflect failure to recognize a rare disease. New surrogate markers are needed to assess disease activity. Glucocorticoids are the cornerstone of treatment and cytotoxic drugs are frequently used as steroid sparing agents.

14th International HLA and Immunogenetics Workshop: report from the reproductive immunology component.

The aim of the study was to investigate whether human leukocyte antigen (HLA) allele sharing between partners or the maternal killer immunoglobulin-like receptor (KIR) repertoire is associated with recurrent spontaneous abortion (RSA) and repeated implantation failure after in vitro fertilization (IVF)/embryo transfer. From a total population of 158 RSA couples, 40 couples with repeated implantation failures (IVF) and 81 control couples, reported by five different laboratories, analysis was performed for (a) HLA sharing in 50 RSA, 31 IVF and 31 control couples, (b) DQA1*0505 sharing/homozygosity among partners in 108 RSA, 40 IVF and 36 control couples, and (c) the women's KIR repertoire in 46 RSA, 26 IVF and 36 control wives. RSA couples were divided into alloimmune aborter (RSAallo) and autoimmune aborter (RSAauto). The results oppose to the suggestion that increased HLA sharing per se or a limited maternal KIR repertoire predisposes to RSA or IVF failure. However, the observation of a slightly higher percentage of DQA1*0505 sharing in the RSAauto and the IVF group needs further investigation. The ratio of inhibitory to activating KIR (actKIR) was slightly lower in RSAallo and IVF women (1.9 vs 2.6 in controls), while in a high percentage of these women, the standard receptors of the KIR A haplotype were combined with actKIR/s of the haplotype B (66.6% and 45.4% vs 20% and 15.3% in RSAauto and control groups). This may suggest a possible involvement of actKIRs in embryo implantation and the maintenance of pregnancy and also requires further investigation.

Lack of the appropriate natural killer cell inhibitory receptors in women with spontaneous abortion.

Previous studies have revealed that women with unexplained recurrent spontaneous abortions have a limited repertoire of inhibitory KI receptors (inhKIRs) and that the inhKIRs they possess do not have specificity for the human leukocyte antigen (HLA)-Cw molecules that would be expressed on trophoblast. We sought to confirm these findings by direct definition of maternal inhKIR and trophoblastic HLA-Cw allotypes on the placental material of spontaneously missed pregnancies. The study included 30 women undergoing vacuum uterine curettage for first-trimester missed pregnancy (group A; n = 15) or for elective termination of normal pregnancy (group C, n = 15). DNA extracted from isolated decidual and trophoblastic cells was used for molecular detection of maternal inhKIRs (2DL1, 2DL2, 2DL3) and fetal HLA-Cw alleles, respectively. The results revealed that in the group of women who experienced abortion, 60% did not have the full repertoire of three inhKIRs (group A vs group C; p = 0.006); that in five of 15 patients (none in the controls), no epitope matching existed between maternal inhKIRs and trophoblastic HLA-Cw alleles (group A vs group C; p = 0.01); and that more cases were found with limited epitope matching (less than three inhKIRs with specificity for fetal HLA-Cw alleles). The results provide additional evidence that in some cases of spontaneous abortions, the women lack the appropriate inhKIRs to interact with the HLA-Cw molecules on trophoblasts and to deliver signals to inhibit natural killer cell activation and protect the embryo.

Minor histocompatibility antigen HA-1 and HPA-5 polymorphisms in HLA-identical related bone marrow transplantation.

The minor histocompatibility antigens (mHags), HA-1 and HPA-5, are immunogenic alloantigens shown to be responsible for graft-versus-host disease (GVHD) in HLA-identical bone marrow transplantation. Both antigens have two known alleles each, resulting in a single amino acid polymorphism. The HA-1H allele encodes histidine, whereas the HA-1R allele encodes arginine. The HPA-5b (Br(a)) allele encodes lysine, whereas the HPA-5a (Br(b)) encodes glutamic acid. In this study, 49 bone marrow transplant recipients and their genetically related HLA-identical donors were evaluated for the presence of HA-1, whereas 39 recipients, different from the abovementioned ones, and their HLA-identical siblings were analyzed for the presence of HPA-5. The frequencies of the two alleles of HA-1 in the recipient population were HA-1R = 0.663 and HA-1H = 0.336. In the donor population, the respective frequencies were 0.704 and 0.296. Seven donors (14.5%) were mismatched with the recipients for HA-1H. In contrast, the frequencies of the two alleles of HPA-5 in the recipient population were HPA-5a = 0.859 and HPA-5b = 0.141; whereas, among donors, they were 0.820 and 0.180, respectively. Five donors (12.8%) were found to be mismatched with their recipients for HPA-5. These results provide insight into the polymorphism of mH antigens based on the study of their frequencies in bone marrow transplant recipients and their genetically HLA-identical siblings, an endeavor that is essential to investigate the presence of HA-1 and HPA-5 mHags.

MHC class I and class II phenotype, gene, and haplotype frequencies in Greeks using molecular typing data.

In the present study, DNA typing for HLA-A, C, B, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, and DPB1 was performed for 246 healthy, unrelated Greek volunteers of 20-59 years of age. Phenotype, genotype frequencies, Hardy-Weinberg equilibrium fit, and 3-locus haplotype frequencies for HLA-A, C, B, HLA-A, B, DRB1, HLA-DRB1, DQA1, DQB1, and HLA-DRB1, DQB1, DPB1 were calculated. Furthermore, linkage disequilibrium, deltas, relative deltas and p-values for significance of the deltas were defined. The population studied is in Hardy-Weinberg equilibrium, and many MHC haplotypes are in linkage disequilibrium. The most frequent specificities were HLA-A*02 (phenotype frequency = 44.3%) followed by HLA-A*24 (27.2%), HLA-B*51 (28.5%), HLA-B*18 (26.8%) and HLA-B*35 (26.4%) and HLA-Cw*04 (30.1%) and HLA-Cw*12 (26.8%). The most frequent MHC class II alleles were HLA-DRB1*1104 (34.1%), HLA-DQB1*0301 (54.5%) and HLA-DPB1*0401 with a phenotype frequency of 59.8%. The most prominent HLA-A, C, B haplotypes were HLA-A*24, Cw*04, B*35, and HLA-A*02, Cw*04, B*35, each of them observed in 21/246 individuals. The most frequent HLA-A, B, DRB1 haplotype was HLA-A*02, B*18, DRB1*1104 seen in 20/246 individuals, while the haplotype HLA-DRB1*1104, DQB1*0301, DPB1*0401 was found in 49/246 individuals. Finally, the haplotype DRB1*1104, DQA1*0501, DQB1*0301 was observed in 83/246 individuals. These results can be used for the estimation of the probability of finding a suitable haplotypically identical related or unrelated stem cell donor for patients of Greek ancestry. In addition, they can be used for HLA and disease association studies, genetic distance studies in the Balkan and Mediterranean area, paternity cases, and matching probability calculations for the optimal allocation of kidneys in Greece.

Systemic scleroderma in Greece: low mortality and strong linkage with HLA-DRB1*1104 allele.

OBJECTIVE: Description of Greek patients with scleroderma with reference to (a) major organ disease, (b) autoantibodies, (c) survival rate, and (d) HLA associations. METHODS: The clinical files of 254 patients were analysed retrospectively and a standardised clinical chart was completed with age at disease onset, sex, date of first and last visit, clinical and serological findings, organs affected, reasons for death, and HLA class II alleles. HLA class II alleles (DRB1, DQA1, DQB1, DPB1) were determined by polymerase chain reaction amplification using oligopeptide probes. DNA was extracted from 98 patients and 130 Greek controls. RESULTS: 124 patients (49%) had limited systemic sclerosis (lSSc), 114 (45%) had diffuse systemic sclerosis (dSSc), and 16 (6%) had overlap syndromes. Patients with dSSc, compared with lSSc, were characterised by a higher prevalence of lung disease (p=0.0011), oesophageal, heart, and peripheral vessel disease (p=0.027, p=0.0025, and p=0.012, respectively). Anticentromere antibodies (ACA) occurred exclusively in lSSc (34%), whereas antibodies to topoisomerase I (anti-topo I) were associated with dSSc (p<0.0001). Anti-topo I were associated with interstitial pulmonary fibrosis, oesophageal and peripheral vessel disease (p=0.028, p=0.012, and p=0.01, respectively). The HLA-DRB1*1104 allele was associated with the disease (p<0.0001) and anti-topo I (p<0.001), whereas it was not associated with ACA serum reactivity (p<0.001). Renal disease occurred in 4% of patients with SSc. The estimated survival probability for this cohort of patients with SSc, four years after the first visit, is 94.8%. CONCLUSION: SSc among Greek subjects has the same pattern of organ disease as in other white populations. However, the prevalence of kidney disease is low. The HLA class II DRB1*1104 allele is associated with the disease, with anti-topo I, and not associated with ACA serum reactivity.

HLA associations of anti-beta2 glycoprotein I response in a Greek cohort with antiphospholipid syndrome and meta-analysis of four ethnic groups.

Using molecular typing, we evaluated the strength of class II HLA associations in 67 Greek patients with antiphospholipid syndrome (APS), 54 of whom had antibodies against beta2-glycoprotein I (beta2GPI), as compared to 246 controls. To further clarify and delineate HLA associations of the beta2GPI response, we combined these data with individual patient data from three other ethnic groups including an additional 74 patients with beta2GPI response and 403 ethnically matched controls of white, African-American, and Mexican-American origin in a formal meta-analysis. The major alleles associated with anti-beta2GPI response are HLA-DQA1*03 (in particular *0301) and the HLA-DRB1*1302-DQB1*0604 haplotype, while protection against developing an anti-beta2GPI response is related primarily to the HLA-DRB1*0101-DQA1*0101 haplotype and the HLA-DRB1*1101 allele. These effects are not significantly heterogeneous across ethnic groups. The previously observed association with HLA-DQB1*0302 may simply reflect linkage disequilibrium with HLA-DQA1*0301 and the previously reported HLA-DQB1*06 effect is limited to HLA-DQB1*0604/0605, while HLA-DQB1*0602 is unlikely to be important. The meta-analysis clearly documents that the anti-beta2GPI response is determined by a few specific class II alleles and haplotypes.