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Item does not contain fulltext BACKGROUND: Pre-transplant donor-specific anti-human leucocyte antigen (HLA) antibodies (DSAs) are associated with impaired kidney graft survival while the clinical relevance of non-donor-specific anti-HLA antibodies (nDSAs) is more controversial. The aim of the present paired kidney graft study was to compare the clinical relevance of DSAs and nDSAs. METHODS: To eliminate donor and era-dependent factors, a post hoc paired kidney graft analysis was performed as part of a Dutch multicentre study evaluating all transplantations between 1995 and 2005 with available pre-transplant serum samples. Anti-HLA antibodies were detected with a Luminex single-antigen bead assay. RESULTS: Among 3237 deceased donor transplantations, we identified 115 recipient pairs receiving a kidney from the same donor with one recipient being DSA positive and the other without anti-HLA antibodies. Patients with pre-transplant DSAs had a significantly lower 10-year death-censored graft survival (55% versus 82%, P=0.0001). We identified 192 pairs with one recipient as nDSA positive (against Class I and/or II) and the other without anti-HLA antibodies. For the patients with nDSAs against either Class I or II, graft survival did not significantly differ compared with patients without anti-HLA antibodies (74% versus 77%, P = 0.79). Only in patients with both nDSAs Class I and II was there a trend towards a lower graft survival (58%, P = 0.06). Lastly, in a small group of 42 recipient pairs, 10-year graft survival in recipients with DSAs was 49% compared with 68% in recipients with nDSAs (P=0.11). CONCLUSION: This paired kidney analysis confirms that the presence of pre-transplant DSAs in deceased donor transplantations is a risk marker for graft loss, whereas nDSAs in general are not associated with a lower graft survival. Subgroup analysis indicated that only in broadly sensitized patients with nDSAs against Class I and II, nDSAs may be a risk marker for graft loss in the long term.

The killer-cell Ig-like receptors (KIRs) play a central role in the immune recognition in infection, pregnancy, and transplantation through their interactions with MHC class I molecules. KIR genes display abundant copy number variation as well as high levels of polymorphism. As a result, it is challenging to characterize this structurally dynamic region. KIR haplotypes have been analyzed in different species using conventional characterization methods, such as Sanger sequencing and Roche/454 pyrosequencing. However, these methods are time-consuming and often failed to define complete haplotypes, or do not reach allele-level resolution. In addition, most analyses were performed on genomic DNA, and thus were lacking substantial information about transcription and its corresponding modifications. In this paper, we present a single-molecule real-time sequencing approach, using Pacific Biosciences Sequel platform to characterize the KIR transcriptomes in human and rhesus macaque (Macaca mulatta) families. This high-resolution approach allowed the identification of novel Mamu-KIR alleles, the extension of reported allele sequences, and the determination of human and macaque KIR haplotypes. In addition, multiple recombinant KIR genes were discovered, all located on contracted haplotypes, which were likely the result of chromosomal rearrangements. The relatively high number of contracted haplotypes discovered might be indicative of selection on small KIR repertoires and/or novel fusion gene products. This next-generation method provides an improved high-resolution characterization of the KIR cluster in humans and macaques, which eventually may aid in a better understanding and interpretation of KIR allele–associated diseases, as well as the immune response in transplantation and reproduction.

Background Complement-fixing antibodies against donor HLA are considered a contraindication for kidney transplant. A modification of the IgG single-antigen bead (SAB) assay allows detection of anti-HLA antibodies that bind C3d. Because early humoral graft rejection is considered to be complement mediated, this SAB-based technique may provide a valuable tool in the pretransplant risk stratification of kidney transplant recipients.Methods Previously, we established that pretransplant donor-specific anti-HLA antibodies (DSAs) are associated with increased risk for long-term graft failure in complement-dependent cytotoxicity crossmatch-negative transplants. In this study, we further characterized the DSA-positive serum samples using the C3d SAB assay.Results Among 567 pretransplant DSA-positive serum samples, 97 (17%) contained at least one C3d-fixing DSA, whereas 470 (83%) had non-C3d-fixing DSA. At 10 years after transplant, patients with C3d-fixing antibodies had a death-censored, covariate-adjusted graft survival of 60%, whereas patients with non-C3d-fixing DSA had a graft survival of 64% (hazard ratio, 1.02; 95% confidence interval, 0.70 to 1.48 for C3d-fixing DSA compared with non-C3d-fixing DSA; P=0.93). Patients without DSA had a 10-year graft survival of 78%.Conclusions The C3d-fixing ability of pretransplant DSA is not associated with increased risk for graft failure.

Two new HLA alleles were identified during routine next generation sequencing.

The clinical significance of non‐HLA antibodies on renal allograft survival is a matter of debate, due to differences in reported results and lack of large‐scale studies incorporating analysis of multiple non‐HLA antibodies simultaneously. We developed a multiplex non‐HLA antibody assay against 14 proteins highly expressed in the kidney. In this study, the presence of pretransplant non‐HLA antibodies was correlated to renal allograft survival in a nationwide cohort of 4770 recipients transplanted between 1995 and 2006. Autoantibodies against Rho GDP‐dissociation inhibitor 2 (ARHGDIB) were significantly associated with graft loss in recipients transplanted with a deceased‐donor kidney (N = 3276) but not in recipients of a living‐donor kidney (N = 1496). At 10 years after deceased‐donor transplantation, recipients with anti‐ARHGDIB antibodies (94/3276 = 2.9%) had a 13% lower death‐censored covariate‐adjusted graft survival compared to the anti‐ARHGDIB‐negative (3182/3276 = 97.1%) population (hazard ratio 1.82; 95% confidence interval, 1.32‐2.53; P = .0003). These antibodies occur independently from donor‐specific anti‐HLA antibodies (DSA) or other non‐HLA antibodies investigated. No significant relations with graft loss were found for the other 13 non‐HLA antibodies. We suggest that pretransplant risk assessment can be improved by measuring anti‐ARHGDIB antibodies in all patients awaiting deceased‐donor transplantation., From a multicenter evaluation of kidney transplants, the authors report that the pretransplant presence of autoantibodies against ARHGDIB are associated with long‐term graft loss in recipients transplanted with a deceased donor kidney, independent from donor‐specific HLA antibodies.

Whereas regular allocation avoids unacceptable mismatches on the donor organ, allocation to highly sensitized patients within the Eurotransplant Acceptable Mismatch (AM) program is based on the patient's HLA phenotype plus acceptable antigens. These are HLA antigens to which the patient never made antibodies, as determined by extensive laboratory testing. AM patients have superior long‐term graft survival compared with highly sensitized patients in regular allocation. Here, we questioned whether the AM program also results in lower rejection rates. From the PROCARE cohort, consisting of all Dutch kidney transplants in 1995‐2005, we selected deceased donor single transplants with a minimum of 1 HLA mismatch and determined the cumulative 6‐month rejection incidence for patients in AM or regular allocation. Additionally, we determined the effect of minimal matching criteria of 1 HLA‐B plus 1 HLA‐DR, or 2 HLA‐DR antigens on rejection incidence. AM patients showed significantly lower rejection rates than highly immunized patients in regular allocation, comparable to nonsensitized patients, independent of other risk factors for rejection. In contrast to highly sensitized patients in regular allocation, minimal matching criteria did not affect rejection rates in AM patients. Allocation based on acceptable antigens leads to relatively low‐risk transplants for highly sensitized patients with rejection rates similar to those of nonimmunized individuals., The authors show that kidney allocation to highly sensitized patients based on proven acceptable HLA antigens results in a significantly lower incidence of rejection episodes when compared to allocation based on the avoidance of unacceptable HLA antigens only.

Supplemental Digital Content is available in the text., Background. There is no consensus in the literature on the interpretation of single-antigen bead positive for a specific HLA antibody. Methods. To inform the debate, we studied the relationship between various single-antigen bead positivity algorithms and the impact of resulting donor-specific HLA antibody (DSA) positivity on long-term kidney graft survival in 3237 deceased-donor transplants. Results. First, we showed that the interassay variability can be greatly reduced when working with signal-to-background ratios instead of absolute median fluorescence intensities (MFIs). Next, we determined pretransplant DSA using various MFI cutoffs, signal-to-background ratios, and combinations thereof. The impact of the various cutoffs was studied by comparing the graft survival between the DSA-positive and DSA-negative groups. We did not observe a strong impact of various cutoff levels on 10-year graft survival. A stronger relationship between the cutoff level and 1-year graft survival for DSA-positive transplants was found when using signal-to-background ratios, most pronounced for the bead of the same HLA locus with lowest MFI taken as background. Conclusions. With respect to pretransplant risk stratification, we propose a signal-to-background ratio-6 (using the bead of the same HLA-locus with lowest MFI as background) cutoff of 15 combined with an MFI cutoff of 500, resulting in 8% and 21% lower 1- and 10-year graft survivals, respectively, for 8% DSA-positive transplants.

Background Few studies have evaluated the effect of different immunosuppressive strategies on long-term kidney transplant outcomes. Moreover, as they were usually based on historical data, it was not possible to account for the presence of pretransplant donor-specific human-leukocyte antigen antibodies (DSA), a currently recognized risk marker for impaired graft survival. The aim of this study was to evaluate to what extent frequently used initial immunosuppressive therapies increase graft survival in immunological low-risk patients. Methods We performed an analysis on the PROCARE cohort, a Dutch multicentre study including all transplantations performed in the Netherlands between 1995 and 2005 with available pretransplant serum (n = 4724). All sera were assessed for the presence of DSA by a luminex single-antigen bead assay. Patients with a previous kidney transplantation, pretransplant DSA or receiving induction therapy were excluded from the analysis. Results Three regimes were used in over 200 patients: cyclosporine (CsA)/prednisolone (Pred) (n = 542), CsA/mycophenolate mofetil (MMF)/Pred (n = 857) and tacrolimus (TAC)/MMF/Pred (n = 811). Covariate-adjusted analysis revealed no significant differences in 10-year death-censored graft survival between patients on TAC/MMF/Pred therapy (79%) compared with patients on CsA/MMF/Pred (82%, P = 0.88) or CsA/Pred (79%, P = 0.21). However, 1-year rejection-free survival censored for death and failure unrelated to rejection was significantly higher for TAC/MMF/Pred (81%) when compared with CsA/MMF/Pred (67%, P Conclusion These results suggest that in immunological low-risk patients excellent long-term kidney graft survival can be achieved irrespective of the type of initial immunosuppressive therapy (CsA or TAC; with or without MMF), despite differences in 1-year rejection-free survival.

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

The presence of donor‐specific anti‐HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long‐term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement‐dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10‐year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10‐year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10‐year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence., Via a retrospective multicenter study, the authors investigate the impact of pretransplant donor‐specific HLA antibodies on long‐term graft survival in deceased and living donor kidney transplantations.

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 4 years. During this period, patients are fully dependent on dialysis, which replaces only partly the renal function, whereas the quality of life is limited. Mortality among 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 among 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 5429 patients transplanted between 1995 and 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. (C) 2014 Published by Elsevier B.V.

Aim The GenDx NGSgo workflow for HLA typing using Next-generation sequencing (NGS) consists of four practical steps: amplification, library preparation, sequencing and data analysis. Out of these four steps the library preparation is the most labor intensive and most interspersed by incubations. To decrease hands-on time and reduce the risk of human error, NGSgo library preparation can be automated from start to finish using a liquid handler workstation. Here we describe the results of the automation of the NGSgo library preparation for Illumina on a Beckman Coulter Biomek 4000 workstation. The workflow was fully automated from amplicon pool to finalized library, making it completely hands-off to accommodate overnight runs and allow the operator to step away from the machine. Methods The Biomek setup included a thermal cycler, 96-well plate magnet, two peltiers devices for cooling 96-well plates and 1.5 ml tubes, a gripper tool, reagent reservoirs and liquid waste disposal. Pipetting was performed by two pipet tools. This setup allowed for flexible processing of 4 to 24 samples per run, with any number of loci. Per run only three boxes of pipetting tips were used for 24 samples. Panels with variable numbers of samples and replicate panels were processed with the Biomek. Subsequently, the pooled libraries were sequenced on a MiSeq system and the resulting data was analyzed with NGSengine software (GenDx). Results MiSeq run parameters were indicative of a high-quality sequencing runs. Read depth, mappability and noise levels of the sequence data presented here showed that the automated workflow resulted in high quality reads and accurate typings. Results from the replicate panel showed a 100% typing match. Conclusions – NGSgo library preparation for 4 to 24 samples can be performed on the Biomek 4000 platform, without manual intervention. – The automated workflow results in high-quality reproducible libraries. – Performing automated NGSgo library preparation as described here reduces hands-on time from 1.5 h to 10 min as compared to manual execution. B. Luiken: 5. Employee; Company/Organization; GenDx. E. Tavkin: 5. Employee; Company/Organization; Beckman Coulter. S. Parakh: 5. Employee; Company/Organization; Beckman Coulter. J. Lu: 5. Employee; Company/Organization; Beckman Coulter. A. Jackson: 5. Employee; Company/Organization; Beckman Coulter. S. van Wageningen: 5. Employee; Company/Organization; GenDx. M. Penning: 5. Employee; Company/Organization; GenDx.

The HLA-B5/35 cross-reacting group (CREG) is a set of closely related antigens including HLA-B35, B51, B52, B53 and B78. The nucleotide sequences of exon 1 through 5 of the B5/35 CREG were determined to assess the level of polymorphism. For exons 2 and 3, the previously described sequence-based typing (SBT) strategy was applied, the nucleotide sequences of exon 1, 4 and 5 were determined by allele-specific sequencing. A total of 225 unrelated individuals were HLA-B typed by heterozygous sequencing of exons 2 and 3. In the B5/35 CREG, 26 different alleles were identified, whereas 63 non-B5/35 CREG alleles were sequenced. The SBT strategy was proven to be reliable and efficient for high resolution typing of the B5/35 CREG. The nucleotide sequences of exon 1, 4 and 5 were determined for the 26 different B5/35 CREG alleles to establish the level of polymorphism. For seven different alleles, of which the exon 1, 4 and 5 sequences were hitherto unknown, the sequences were elucidated and in agreement with the known B5/35 sequences. Nineteen HLA-B5/35 CREG alleles with previously published exon 1, 4 and 5 sequences were sequenced in at least two individuals. Three new alleles were identified. The first, B*5204, showed a difference at position 200 compared to B*52011, which was previously considered a conserved position. The other two alleles, B*3542 and B*51015, showed exon 2 and 3 sequences identical to B*35011 and B*51011, but differences in exons 1 and 4, respectively. B*3542 had differences at position 25 and 72 and B*51015 showed a difference at position 636. More polymorphism might be present outside exons 2 and 3 than previously thought.

HLA-B*27 is known to be associated with ankylosing spondylitis and several methods have been applied to determine its presence or absence. In this report two molecular methods were used for detection of B*27. The polymerase chain reaction sequence-specific primer (PCR-SSP) method was performed to detect the presence or absence of B*27, whereas the sequence-based typing method (SBT) was used to identify the B*27 subtype. The PCR-SSP method used to detect B*27 was updated to enable the detection of all B*27 alleles. The typing results obtained by this method were compared with the serological typings of 262 individuals. Fifty of them were found to be B*27 positive by PCR-SSP and 46 also showed positive serological reactions with B27-specific sera. The four discrepancies were the result of the presence of B*2712 in three individuals and B*2715 in one individual; both alleles showed no serological reactions with B27-specific antisera. With SBT the sequences of exons 1 through 4 were determined to unequivocally assign the B*27 alleles. Eleven different subtypes were detected in 78 individuals, including three new B*27 alleles: B*27054, B*2715 and B*2717. The allele B*27054 showed an allelic drop out when exon 3 was amplified. Three differences with B*27052 were demonstrated; one in exon 1, one in intron 1 and one in intron 2, the latter being responsible for the allelic drop out. The B*2715 allele was serologically not detectable with several B27-specific sera, but showed Bw4-positive reactions. The sequence of B*2715 showed two mismatches with B*2704. The sequence of B*2717 showed one mismatch with B*27052 at position 248 (A-->T), which was considered to be a conserved position in all B alleles.

Aim Individual HLA mismatches may have differential effects on graft survival after kidney transplantation. Therefore, there is a need for a reliable tool to define permissible HLA mismatches in kidney transplantation. We previously demonstrated that donor-derived Predicted Indirectly ReCognizable HLA epitopes presented by recipient HLA class-II (PIRCHE-II) play a role in de novo donor-specific HLA antibody formation after kidney transplantation. Moreover, we also showed that PIRCHE-II is involved in HLA antibody formation after pregnancy. In the present Dutch multi-center study we evaluated the possible association between PIRCHE-II and kidney graft failure in 2918 donor-recipient couples that were transplanted between 1995 and 2005. Methods For all donors-recipients couples, PIRCHE-II numbers were determined and related to graft survival in both univariate and multivariable analyses. Results Adjusted for confounders, the natural logarithm of PIRCHE-II was associated with a higher risk for graft failure (HR:1.13, 95% CI:1.04–1.23, p = 0.003). Univariately analyzed, patients with low PIRCHE-II numbers had a better 10-years graft survival than patients with higher PIRCHE-II numbers (p = 0.006; PIRCHE-II strata: Conclusions Our data suggest that the PIRCHE-II algorithm is a valuable tool to discriminate between permissible HLA mismatches and high-risk HLA mismatches in kidney transplantation. Inclusion of PIRCHE-II in donor-selection criteria may eventually lead to an improved kidney graft survival.

Molecular typing methods of HLA-B, like sequence-specific oligonucleotide hybridization and sequence-based typing, are based on gene-specific amplifications of exons 2 and 3 followed by probe hybridization or sequence determination. The necessary gene-specific amplification primers are often located in rather conserved regions of the introns. In several of these procedures HLA-B*73 was not amplified, resulting in drop-out of the allele. To investigate the reason for the allelic drop-out, the sequences of introns 1, 2 and 3 of HLA-B*7301 were determined. Comparison of the intron sequence of B*7301 with other HLA-B and HLA-C alleles revealed several remarkable features. The overall sequence resembles the sequence of other HLA-B alleles, although 35 differences were found with a consensus intron sequence. The insertions and deletions shown in intron 2 of B*73 were strikingly similar with the sequences of the HLA-C alleles, as was the 5′ end of intron 3. Furthermore, a unique deletion was observed in the middle of intron 3, not noticed in other HLA-B or C alleles. The HLA-B-specific primers, widely used for sequence-specific oligonucleotide hybridization and sequence-based typing purposes, showed mismatches with the B*73 intron sequences, causing the allelic drop-out. Correct amplification of complete exons 2 and 3 of B*7301 was enabled by the design of new primers in intron 2 and 3.

Killer immunoglobulin-like receptors (KIRs) are involved in the regulation of natural killer cell cytotoxicity. Within the human genome seventeen KIR genes are present, which all contain a large number of allelic variants. The high level of homology among KIR genes has hampered KIR genotyping in larger cohorts, and determination of gene copy number variation (CNV) has been difficult. We have designed a multiplex ligation-dependent probe amplification (MLPA) technique for genotyping and CNV determination in one single assay and validated the results by next-generation sequencing and with a KIR gene-specific short tandem repeat assay. In this way, we demonstrate in a cohort of 120 individuals a high level of CNV for all KIR genes except for the framework genes KIR3DL3 and KIR3DL2. Application of our MLPA assay in segregation analyses of families from the Centre d'Etude du Polymorphisme Humaine, previously KIR-genotyped by classical techniques, confirmed an earlier reported duplication and resulted in the identification of a novel duplication event in one of these families. In summary, our KIR MLPA assay allows rapid and accurate KIR genotyping and CNV detection, thus rendering improved transplantation programs and oncology treatment feasible, and enables more detailed studies on the role of KIRs in human (auto)immunity and infectious disease.

The new A*03:134 differs from A*03:01:01:01 by one amino acid change at codon 264.

In humans, the region configurations DR1, DR8, DR51, DR52 and DR53 are known to display copy number as well as allelic variation, rendering high resolution typing of HLA-DRB haplotypes cumbersome. Advantage was taken of microsatellite D6S2878, present in all DRB genes/pseudogenes with an intact exon 2-intron 2 segment. This DRB-STR is highly polymorphic in composition and length. Recently, it was proven that all exon 2 sequences could be linked to a certain DRB-STR that segregates with the respective DRB allele. Because haplotypes show differential copy numbers and compositions of exon 2- positive DRB genes/pseudogenes, unique DRB-STR patterns could be described that appear to be specific for a particular DRB haplotype. The aim of this workshop project was to approve and to qualify this simple typing protocol in a larger panel covering different European populations. All participants succeeded in correctly defining the DRB-STR amplicons varying from 135 to 222 base pair (bp) lengths. The panel of 101 samples covered 50 DRB alleles distributed over 37 different haplotypes as defined by exon 2 sequence-based typing. These haplotypes could be refined into 105 haplotypes by DRB-STR typing. Thus, discrimination of exon 2-identical DRB alleles was feasible, as well as the exact description of three different crossing-over events that resulted in the generation of hybrid DR region configurations. This typing procedure appears to be a quick and highly robust technique that can easily be performed by different laboratories, even without experience in microsatellite typing ; thus, it is suitable for a variety of researchers in diverse research areas.

Two new human leukocyte antigen (HLA) class II alleles were identified during routine sequence-based typing.

More than 590 human leukocyte antigen (HLA)-B alleles have been identified by sequence analysis. Although the polymorphic exon 2 and 3 sequences of all HLA-B alleles are described, the sequences of the other exons of a number of infrequent B-alleles are unknown. In this study, the exon 1, 4, and 5 sequences of 39 different HLA-B alleles were elucidated by allele-specific sequencing. Overall, these exon sequences showed identity with the majority of the known sequences from the corresponding allele groups, except for four alleles B*4010, B*4415, B*4416, and B*5606. The exon 1 sequence of B*4010 had nucleotide differences with all B*40 alleles, but was identical to the B*54, *55, *56, and *59 allele groups. B*4416 differed from B*440201 at position 988, which was previously considered a conserved position. B*4415 showed exon 1, 4, and 5 sequences deviating from the other B*44 alleles, but identical to B*4501. The exon 1 and 4 sequences of B*5606 differed from other B*56 alleles, but were in complete agreement with B*7801. The deviating exon sequences of B*4415 and B*5606 confirmed the evolutionary origin of these alleles suggested by the sequences of exons 2 and 3. The polymorphism observed in exons 1, 4, and 5 merely reflects the lineage-specificity of HLA-B.

An unusual haplotype was detected in a family of a caucasian transplant patient. Human leukocyte antigen (HLA) analysis of the family demonstrated the absence of HLA-A on one of the haplotypes present in two family members. One was serologically typed A24, the other A2. Because they had one haplotype in common, the HLA-A allele of the shared haplotype was supposed to be a null allele. Different molecular typing methods identified only one allele in both individuals. The results suggest a deletion of the complete HLA-A gene or a major part of it. For confirmation, microsatellite analysis of the HLA-A region was performed with six microsatellite markers. Both family members were heterozygous for all markers, and a deletion of HLA-A could not be proven. Fluorescent in situ hybridization (FISH) was performed with cosmid and PAC probes encompassing the HLA-A gene. Both probes demonstrated an identical normal distribution pattern for diploid results. The absence of any serologic and molecular reaction with the results of the microsatellite and FISH analysis make a deletion of a narrow region, encompassing the HLA-A gene, the most plausible explanation.

The strategy for sequencing human leukocyte antigen (HLA)-A was based on separate amplification of exons 2 and 3, followed by forward and reverse heterozygous sequencing of the alleles. Validation of the method was obtained by sequencing 11 individuals carrying alleles from all different HLA-A allele groups, except *43. All alleles could be correctly identified except A*3401. Unexpected polymorphic positions were identified in exon 3, even in individuals homozygous for A*3401. In addition, the pseudogene HLA-COQ or HLA-DEL linked to A*3401 was coamplified and sequenced. The problem was solved by using different amplification primers for exon 3 with mismatches for the two pseudogenes. A total of 252 unrelated individuals with at least one allele belonging to the A10 or A19 group were typed for HLA-A by this strategy. Ten different alleles were identified in the A10 group and 14 in the A19 group. As second allele a further 30 different subtypes from all different groups were sequenced. In 21 individuals, sequencing exon 1 was necessary to distinguish A*7401 from A*7402. The sequencing strategy, with separate amplification of the exons, has proven to be a robust method, resulting in reliable and efficient high-resolution HLA-A typing.

The elucidation of the sequences of human leukocyte antigen-B (HLA-B)-exons 1 through 5 has led to an increase of ambiguities with alleles having identical exon 2 and 3 sequences, but differences in other exons. At the moment, 26 HLA-B alleles show such ambiguities which can be resolved by sequencing the exons in which the differences are located. Here we report a sequence-based typing (SBT) strategy for heterozygous sequencing of exons 1, 4, and 5, in addition to the previously described exons 2 and 3. The strategy was validated against a panel of 25 individuals, carrying HLA-B alleles from 33 different allele groups. Correct assignment of all HLA-B alleles was obtained for exons 1 through 5. In addition, the SBT protocol was used to resolve ambiguities in 50 individuals. The ambiguous combinations studied were B*0705/06, B*0801/19N, B*1512/19, B*180101/17N, B*270502/13/0504, B*350101/42/40N, B*390101/0103, B*400102/0101, B*440201/19N/27, and B*510101/11N/0105/30/32. In all cases, sequencing revealed the first allele to be present, except for three individuals with B*07. One of them typed B*0705; the other two were B*0706. The described SBT protocol for sequencing exons 1, 4, and 5 is a valuable tool for resolving ambiguities of HLA-B alleles with differences in these exons, as well as for studying the polymorphism of HLA-B outside exons 2 and 3.