[Show abstract][Hide abstract] ABSTRACT: Kidney transplantation is the best treatment option for patients with end-stage renal failure. At present, approximately 800 Dutch patients are registered on the active waiting list of Eurotransplant. The waiting time in the Netherlands for a kidney from a deceased donor is on average between 3 and 4years. During this period, patients are fully dependent on dialysis, which replaces only partly the renal function, whereas the quality of life is limited. Mortality amongst patients on the waiting list is high. In order to increase the number of kidney donors, several initiatives have been undertaken by the Dutch Kidney Foundation including national calls for donor registration and providing information on organ donation and kidney transplantation. The aim of the national PROCARE consortium is to develop improved matching algorithms that will lead to a prolonged survival of transplanted donor kidneys and a reduced HLA immunization. The latter will positively affect the waiting time for a retransplantation. The present algorithm for allocation is amongst others based on matching for HLA antigens, which were originally defined by antibodies using serological typing techniques. However, several studies suggest that this algorithm needs adaptation and that other immune parameters which are currently not included may assist in improving graft survival rates. We will employ a multicenter-based evaluation on 5.429 patients transplanted between 1995-2005 in the Netherlands. The association between key clinical endpoints and selected laboratory defined parameters will be examined, including Luminex-defined HLA antibody specificities, T and B cell epitopes recognized on the mismatched HLA antigens, non-HLA antibodies, and also polymorphisms in complement and Fc receptors functionally associated with effector functions of anti-graft antibodies. From these data, key parameters determining the success of kidney transplantation will be identified which will lead to the identification of additional parameters to be included in future matching algorithms aiming to extend survival of transplanted kidneys and to diminish HLA immunization. Computer simulation studies will reveal the number of patients having a direct benefit from improved matching, the effect on shortening of the waiting list, and the decrease in waiting time.
[Show abstract][Hide abstract] ABSTRACT: Our understanding of the immunological processes influencing the clinical outcome after kidney transplantation has advanced majorly over the last few decades. However, many factors still restrict graft and patient survival. Within the Maastricht transplant centre we have successfully implemented an alternative immunosuppressive regimen involving Tacrolimus monotherapy in order to minimize the adverse effects associated with long-term use of immunosuppressive drugs. This clinical development has an impact on pre-transplant risk stratification which requires that patients are closely monitored immunologically. In this review we will elaborate on our strategy regarding the analysis of epitopes in HLA-DQ and HLA-DP molecules. In this respect we have also looked at the immunodominance of certain epitopes by assessing their structural localisation, conformation and physiochemical properties.
[Show abstract][Hide abstract] ABSTRACT: The functional relevance of human leukocyte antigen (HLA) class I allele polymorphism beyond exons 2 and 3 is difficult to address because more than 70% of the HLA class I alleles are defined by exons 2 and 3 sequences only. For routine application on clinical samples we improved and validated the HLA sequence-based typing (SBT) approach based on RNA templates, using either a single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications, with three forward and three reverse sequencing reactions for full length sequencing. Locus-specific HLA typing with RNA SBT of a reference panel, representing the major antigen groups, showed identical results compared to DNA SBT typing. Alleles encountered with unknown exons in the IMGT/HLA database and three samples, two with Null and one with a Low expressed allele, have been addressed by the group-specific RNA SBT approach to obtain full length coding sequences. This RNA SBT approach has proven its value in our routine full length definition of alleles.
[Show abstract][Hide abstract] ABSTRACT: Alloreactivity to HLA-DP molecules, class II heterodimers of an oligomorphic alpha and a polymorphic beta chain, is increasingly being studied due to its relevance in clinical transplantation. We hypothesized that not only polymorphisms in the peptide binding groove encoded by exon 2 of HLA-DPB1, but also in other regions of the molecule and the alpha chain, could play a role in CD4+ T cell allorecognition. To test this possibility, we comparatively investigated CD4+ T cell allorecognition, measured by upregulation of the activation marker CD137, against HLA-DPB1*13:01, *05:01, *03:01, *17:01 or their allele counter parts DPB1*107:01, *135:01, *104:01, *131:01, with identical exon 2 sequences but polymorphism in exons 1, 3 or 4, in the context of different HLA-DPA1 (DPA1) polymorphisms (DPA1*01:03 and *02:01). No significant differences in CD4+ T cell allorecognition levels could be demonstrated for any of the beyond exon 2 DPB1 variants studied. Interestingly, however, the mean fold change in CD4+ CD137+ cells was significantly higher when the target shared at least one DPA1 allele with the allogeneic stimulator, compared to a distinct DPA1 background (1.65 vs 0.23, P<0.005). Structural homology modeling suggested specific amino acid residues in the alpha chain, in particular position 31, to impact CD4+ T cell allorecognition of HLA-DP. Our data argue against a significant role of beyond exon 2 DPB1 polymorphisms for T cell alloreactivity, but show relevance of DPA1 polymorphism in this mechanism. These new findings impact HLA matching strategies in unrelated stem cell transplantation.
[Show abstract][Hide abstract] ABSTRACT: Genetic polymorphism of human leukocyte antigen (HLA)-DPA1 and -DPB1 loci was studied in 154 unrelated individuals from Guadeloupe, an archipelago of five islands located in the Carribean Sea. Thirty different DPB1 and eight different DPA1 alleles were observed with a heterozygosity index of 0.87 and 0.78, respectively. This high degree of heterozygosity corresponds with those found in African populations. The DPB1* 01:01:01 allele was most frequent (0.260), followed by 02:01:02 (0.143) and 04:01:01 (0.127). The DPA1 alleles 01:03 (0.380), 02:01 (0.302), 02:02 (0.175) and 03:01 (0.123) were identified in >35 individuals each, whereas 01:04, 01:05 and 04:01 were present only once. Haplotype estimations revealed the presence of 39 different haplotypes, with DPB1*01:01:01-DPA1*02:02 and DPB1*02:01:02-DPA1*01:03 as the most frequent (0.143 and 0.140, respectively). A striking difference was observed in DPB1/DPA1 associations between DPB1*04:02 and *105:01, that have identical exon 2 sequences. DPB1*04:02 was exclusively associated with DPA1*01:03, whereas DPB1*105:01 was present with DPA1*03:01, *03:02 or *04:01. This implies that the DP molecules are actually different, and this difference is relevant to consider in studies on the function of HLA-DP molecules in transplantation. Overall, HLA-DPA1 and DPB1 allele frequencies and haplotypes of the population of Guadeloupe were most similar to African populations, with characteristic alleles and haplotypes that bespeaks the admixture with other ethnicities.
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is classically attributed to the dysfunction of the single CF transmembrane conductance regulator gene. The incidence of human leukocyte antigen (HLA) polymorphisms in different CF-associated diseases raises the question of an unequal distribution of HLA genotypes in CF. This study aimed to evaluate HLA gene frequencies and possible associations in CF patients compared with a control population. Frequencies of HLA-DRB1, HLA-DQA1 and HLA-DQB1, performed by intermediate resolution typing using Luminex sequence-specific oligonucleotide, and epitope counts were similar in 340 CF patients when compared with 400 control subjects. In conclusion, HLA-DRB1, -DQA1 and -DQB1 do not seem to influence susceptibility to CF. Whether HLA plays a role in the severity of CF disease needs to be investigated.
[Show abstract][Hide abstract] ABSTRACT: Matching for HLA at the allele level is crucial for stem cell transplantation. The golden standard approach for allele definition of full gene polymorphism, the so-called high-resolution HLA typing, is sequence-based typing (SBT). Although the majority of the polymorphism for class I is located in exons 2 and 3 and for class II in exon 2, for allele definition it is necessary to unravel the complete coding and intron sequences leading to an ultrahigh HLA typing resolution at the allele level, i.e., a full-length gene polymorphism identification.This chapter describes our recently developed SBT method for HLA-A, -B, -C, and -DQB1, that is based on full-length hemizygous Sanger sequencing of the alleles, separated by group-specific amplification using the low-resolution typing result as reference starting point. Group-specific amplification has already been established for DRB. This method enables a cost-efficient, user-friendly SBT approach resulting in a timely unambiguous HLA typing to an ultrahigh resolution level with minimal hands-on time.
[Show abstract][Hide abstract] ABSTRACT: We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of ~140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.
[Show abstract][Hide abstract] ABSTRACT: Despite DP antigens have been shown to be stimulators of the mixed lymphocyte reaction, human leukocyte antigen-DPB1 is not considered in the matching criteria for hematopoietic stem cell transplantation (HSCT). The role of DPB1 matching in HSCT remains inconclusive because of contradictory findings in different studies. The concept of permissible and non-permissible mismatches might clarify these contradictory results. Although several groups have attempted to identify immunogenic epitopes in exon 2 to establish permissive and non-permissive allele groups, the direct correlation between individual exon 2 amino acids and epitopes with DPB1 immunogenicity is still not evident. We hypothesize that polymorphism within the entire molecule, including polymorphic variability in different ethnic groups, is crucial to unravel the function of DPB1 polymorphism. Using an RNA-based approach, we sequenced all frequent and available non-frequent DPB1 alleles full length from 148 samples representing 28 different DPB1 alleles from either Black, Caucasian, or Oriental origin. We identified various DPB1 alleles with, in addition to the exon 2 polymorphism, polymorphisms in exons 1, 3, 4, and 5. Based on this polymorphism outside exon 2, we defined one new allele. Two alleles with identical exon 2 polymorphism but differing outside exon 2 were identified in individuals of different ethnic groups. As T cell binding is not restricted to the polymorphic groove and polymorphism in the β2 domain of the DP molecule affects CD4 interaction, full-length polymorphism should be considered to determine immunogenicity. Eventually, this knowledge will provide new insights in the classification of DPB1 polymorphism and more importantly will add new perspectives to the concept of permissiveness in transplantation.
[Show abstract][Hide abstract] ABSTRACT: Human leukocyte antigen-E (HLA-E) is a non-classical HLA class I gene that shows a limited degree of polymorphism compared to the classical HLA genes. The HLA-E molecule can bind peptides derived from the leader sequence of various HLA class I alleles and some viral homologues, including CMV. The HLA-E peptide complex can act as a ligand for the CD94/NKG2 receptors expressed on the surface of natural killer cells and T cell subsets. Differences in expression levels between the different HLA-E alleles have been reported and a role for HLA-E polymorphism in stem cell transplantation has been postulated. This chapter focuses on routine technologies for HLA-E typing: the sequence-specific primer-PCR method that uses sequence-specific primers, the PCR sequence-specific oligonucleotides Luminex method, using sequence-specific probes attached to beads and the sequencing-based typing method, where sequencing of the alleles is performed.
[Show abstract][Hide abstract] ABSTRACT: The new HLA-B*15:220 allele shows a single-nucleotide substitution in exon 1 at position 47 (C>T) when compared to its closest allele HLA-B*15:03:01, resulting in an amino acid substitution from Ala to Val in the signal peptide at codon -9.
[Show abstract][Hide abstract] ABSTRACT: The aims of this study were to assess the association of BTNL2G16071A with the course of pulmonary sarcoidosis and to verify the association with disease predisposition. In addition, the linkage between BTNL2G16071A and certain human leukocyte antigen (HLA)-DRB1/DQB1 types was investigated. In a retrospective case-control study BTNL2G16071A, HLA-DQB1, and HLA-DRB1 were typed in 632 sarcoidosis patients. These patients were classified into 304 patients with persistent sarcoidosis and 328 patients with nonpersistent sarcoidosis. The BTNL2 16071A variant allele was present significantly more often in patients with persistent disease (92.4%; 281/304) compared with patients demonstrating a nonpersistent course (86.6%; 284/328; odds ratio (OR) = 1.89 with 95% confidence interval (95% CI) 1.11-3.22). Furthermore, BTNL2 16071A variant allele carriers have an increased risk (OR = 1.85, 95% CI 1.19-2.88) of developing sarcoidosis. Moreover, the strong linkage between variant allele and HLA-DRB1*15 presence (OR = 8.43, 95% CI 3.02-23.5) was confirmed. The presence of a BTNL2G16071A variant allele almost doubles the risk of progressing to persistent pulmonary sarcoidosis in addition to increasing the risk of developing sarcoidosis. Presumably, these increased risks are caused by the strong linkage between BTNL2G16071A and DRB1*15. The choice between determining BTNL2G16071A SNP or the HLA-DRB1 type depends on the ability and/or availability to perform either test.
Human immunology 01/2011; 72(4):342-7. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An unusual haplotype without a detectable human leukocyte antigen (HLA)-A allele by serologic or molecular typing methods segregates in a Caucasian family. Microsatellite analysis and fluorescence in situ hybridization implicated that the deletion encompasses a narrow region. To identify the deleted region, five different fragments in close proximity to HLA-A, known to be highly polymorphic, were amplified and sequenced. The presence of heterozygous sequences in all five fragments of the individuals carrying the haplotype with the HLA-A deletion, indicates that the fragments are not involved in the deletion. Therefore, the 5' primer from the fragment closest to the centromeric side of HLA-A was combined with the 3' primer closest to the telomeric side encompassing an 11-kb region. Sequencing revealed that a deletion of 4089 bp was present, located upstream of HLA-A, including exons and introns 1-3 of the HLA gene. Sequence information of the 3' part of HLA-A, downstream the deletion, identified that the deleted allele originates from an A*24 allele. Although different repeat sequences are present in the region both inside and outside the deletion, no evidence points to a retrotransposon mechanism. The detected partial deletion of HLA-A turns this functional gene into a pseudogene.
Human immunology 12/2010; 71(12):1197-202. · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human leukocyte antigen (HLA)-DP is considered a target for humoral immune response in clinical transplantation. This study analyses the incidence of HLA-DP antibodies in renal patients. Development and epitope specificity of donor-specific antibodies (DSA) and non-DSA (NDSA) were examined. Pre- and posttransplant sera of 338 patients were screened for HLA-DP antibodies using the luminex single antigen assay. Positive patients, partners and/or kidney donors were HLA-DP typed by sequence-specific oligonucleotides. Potential epitopes were mapped by comparing the amino acid sequences of HLA-DP hypervariable regions (HVR) A-F of recipient, partner and/or donor. Specificities in the sera were aligned to deduce the HVR motif responsible for the antibodies. HLA-DP antibodies were detected in 14% of the patients (48/338). Before transplantation, the antibodies were shown in 23% (10 females and 1 male) and 77% were found after transplantation (30 in patients after the first, 7 after the second graft). Specificities were never restricted to individual mismatched antigens; broad HLA-DP sensitization was found as a rule. A single HVR mismatch was present in 80% of the DSA and in 79% of the NDSA. No HLA-DPA specific antibodies were found. Our findings confirm that HLA-DP antibodies are specific for epitopes shared by different HLA-DP antigens, indicating that only a restricted number of mismatched epitopes are recognized by the recipients immune system. Matching for immunogenic HLA-DP epitopes for renal transplantation seems to be functionally more relevant than classical matching at the allelic level.
[Show abstract][Hide abstract] ABSTRACT: This study was designed to evaluate the relationship between the presence of tumor necrosis factor (TNF) polymorphisms, human leukocyte antigen (HLA)-DRB1*03 linkage and the prognosis of sarcoidosis. In a retrospective case-control study, TNF-alpha G-308A, TNF-alpha G-238A, lymphotoxin-alpha (LTA) and HLA-DRB1*03 were genotyped in 625 sarcoidosis patients. These patients were classified into 298 patients with persistent disease and 327 patients with non-persistent disease using chest X-ray (CXR) appearances and lung function parameters after at least 2 years of follow-up. The TNF-alpha-308A variant allele was observed in 25.5% of patients with persistent disease compared with 44.0% of patients with non-persistent disease. The corresponding odds ratio (OR) was 0.43 with a 95% confidence interval (CI) of 0.30-0.61. A strong linkage was found between TNF-alpha G-308A and HLA-DRB1*03 (OR = 0.03, 95% CI: 0.02-0.05). For TNF-alpha G-238A and LTA NcoI A252G, there were no statistically significant differences in the distribution of genotypes between the groups with and without persistent disease. The data indicate that presence of a TNF-alpha-308A variant allele and HLA-DRB1*03 were associated with a favorable prognosis. Because of the strong linkage between TNF-alpha G-308A and HLA-DRB1*03, genotyping of one simple and less expensive TNF-alpha single nucleotide polymorphism can be used to predict the prognosis of pulmonary sarcoidosis in clinical practice.