HLA-DP antibodies before and after renal transplantation.

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.

Clinical relevance of Luminex donor-specific crossmatches: data from 165 renal transplants.

The clinical significance of the presence of donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA) prior to renal transplantation detected solely by solid-phase techniques remains unclear. This study was designed to determine the clinical relevance of the recently introduced bead-based Luminex donor-specific crossmatch (LumXm). A group of 165 patients transplanted between 1997 and 2001 were tested. Of 165 recipients transplanted with a negative complement-dependent cytotoxicity crossmatch, 32 proved to have a positive Luminex crossmatch. Sixteen were positive for class I, 15 were positive for class II, 1 was both class I and II positive and 133 recipients were negative. Acute rejection (AR)-free survival for all recipients was 77%, and there was no difference in AR-free survival between LumXm-positive and LumXm-negative recipients. Overall graft survival after a median follow-up time of 8 years was 56%. Recipients with a positive class I LumXm had worse long-term graft survival (P = 0.006). In recipients with a positive class I LumXm, 5-year graft survival was 41% vs 70% in negative patients and 10-year graft survival was 27% vs 56%. Positivity for class II LumXm was not a significant risk factor for graft failure (P = 0.7). In conclusion, pretransplant DSA detected by the LumXm had no impact on AR episodes. Class II LumXm positivity proved no significant risk factor for graft failure, but the value of the class II LumXm is questionable. A positive class I LumXm resulted in worse long-term graft survival compared with a negative one.

Luminex donor-specific crossmatches.

In Luminex bead-based screening assays, color-coded microspheres coated with human leukocyte antigens (HLA) are used to identify both complement-binding and non-complement-binding HLA class I and II antibodies in recipient sera. Many laboratories rely on their specificity detection and use the information obtained for allocation of donor organs. A donor-specific crossmatch in the Luminex technique (LumXm) is for that reason desirable. A LumXm, in which the actual donor HLA are coated onto specific capture beads, was tested for 88 pre- and posttransplant sera of 18 recipients. The results were compared with previously published flow cytometric crossmatch (FCXm) results for the same donor-recipient combinations. All sera were also examined by Luminex single antigen (SA) tests. Class I LumXm detected 24 of 27 T-cell positive FCXm (89%) and class II 15 of 22 B-cell positive FCXm (68%). Sensitivity of LumXm for class I and II was 89% and 68% and specificity was 98% and 97%, respectively. Discrepant LumXm results were obtained in 13 sera of nine patients (15%). In general, based on SA testing, FCXm showed false-positive results for class I and LumXm gave false-negative and positive results for class II. The LumXm test was proven not to react with recipient sera containing DQ antibodies only, also DP detection was insufficient. The validity of the LumXm has been shown for class I, but its value for class II is uncertain. HLA-DR is most probably correctly identified, the validity for DQ and DP is doubtful.

Reanalysis of sequence-based HLA-A, -B and -Cw typings: how ambiguous is today's SBT typing tomorrow.

The permanently increasing number of human leukocyte antigen (HLA)-alleles and the growing list of ambiguities require continuous updating of high-resolution HLA typing results. Two different kinds of ambiguities exist: the first, when two or more allele combinations have identical heterozygous sequences, and the second, when differences are located outside the analyzed region. The number of HLA-A, B and C alleles recognized in 1999 was almost tripled in 2006. Two hundred individuals, sequence-based typing (SBT) typed in the period from 1999 to 2002, were reanalyzed using the 2006 database. A final allele typing result of at least four digits was obtained for HLA-A, -B and -C by heterozygous sequencing of exons 2 and 3 and, if necessary, additional exons and/or allele-specific sequencing. Storage of the individual sequences in a specially developed database enabled reanalysis with all present and future HLA releases. In the 5-year period HLA-A, -B and -C typing results became ambiguous in 37%, 46% and 41% of the cases. Most were because of differences outside the analyzed region; ambiguities because of different allele combinations with identical heterozygous sequences were present in 7%, 8% and 13% of the HLA-A, -B and -C typings. These results indicate that within 5 years, approximately half of the HLA SBT typings become ambiguous.

HLA class II amino acid epitopes as susceptibility markers of sarcoidosis.

Sarcoidosis is a multisystemic disorder of unknown etiology, affecting primarily the lung and characterized by epithelioid granulomas. Disease association studies showed human leukocyte antigen (HLA) class II to be related to sarcoidosis. Initially, we studied the association of sarcoidosis with DQB1, and in the present study, we evaluated all amino acid variants of the HLA-DPB1, -DQB1, -DRB1, -DRB3, -DRB4 and -DRB5 genes to identify possible polymorphisms associated with the disease. Patients and controls were typed for class II genes to the allele level by sequence-based typing. Multiple logistic regression models showed DRAla71 and DQPhe9 to be independently associated with the disease. Subdivision of patients according to their radiographic stage resulted in identification of DRArg74 as independent associated residue in the RS I group, whereas DRAla71 and DQTyr30 were associated with RS II-IV groups. Polymorphic residues specifically associated with sarcoidosis shed new light on the characteristics of sarcoidosis-triggered peptides. Overall, pocket 9 of DQ and pocket 4 of DR seem to be the most important areas involved in the association with sarcoidosis.

Sequence-based typing of HLA-DQA1: comprehensive approach showed molecular heterogeneity.

Within the human leukocyte antigen-DQA1 workshop project the level of molecular heterogeneity of the DQA1 gene was investigated. An improved sequence-based typing protocol was used, enabling analysis of the complete coding sequence, comprising exons 1-4. The participating laboratories implemented the amplification and sequencing primers in their own sequence-based typing approach. The method proved to be sufficiently robust to handle the differences in protocols. All reference samples used for validation were correctly typed for DQA1 by all participating laboratories. Three different populations with a total of 736 individuals were investigated: a population of Korean origin (n= 467), a British Caucasian (n= 114), and a Dutch Caucasian (n= 155) population. Sixteen of the known 28 DQA1 alleles were detected and six new alleles were identified. All novel alleles showed a nucleotide substitution outside exon 2. Comparison of the calculated allele frequencies revealed major differences between the Korean and the Caucasian populations but also between Dutch and British Caucasians. A tight association between DQA1 and DRB1/DQB1 alleles was observed in all three populations.

A novel HLA-A*11 allele, A*1118, identified by sequence-based typing.

The novel allele A*1118 shows three nucleotide differences with A*110101, resulting in three amino acid changes at positions 70, 74 and 90 of the mature protein.

Identification of the new allele B*3556 and extension of the sequences of B*4420 and B*4427.

A new B*35 allele, B*3556, was identified in a Caucasian individual. Direct sequencing of exons 2 and 3 revealed that B*3556 was identical to B*3503, except for a nucleotide substitution from G to A at position 302, which causes a change from AGC to AAC at codon 101. This results in an amino acid change from Ser to Asn at position 77 of the mature protein. Two B*44 alleles, B*4420 and B*4427, were detected in a liver recipient and in an unrelated bone marrow donor, respectively, and their sequences were confirmed. The sequences of exons 1,4 and 5 were also elucidated; for B*4420, these exons proved to be identical to those of B*440201. The sequence of exon 5 of B*4427 showed three mismatches with that of B*440201, implicating that it probably arose from B*440201 by allele conversion with an allele of the B*15, 45, 46, 49 or 50 allele group. Elucidation of the sequence of introns 3 and 4 indicated that the breakpoint for allele conversion has to be located in intron 3 or exon 4.

Allelic polymorphism in introns 1 and 2 of the HLA-DQA1 gene.

Human leukocyte antigen (HLA) class II antigens are highly polymorphic membrane glycoproteins, encoded by the A and B genes of DR, DQ, and DP. The polymorphism is mainly located in exon 2, with the exception of DQA1. Of the 27 DQA1 alleles presently known, 18 cannot be identified on the basis of exon 2 alone, but need additional information from the other exons. DQA1 has been reported to be the most ancient class II gene. For evolutionary comparison and to assess the degree of polymorphism outside the exons, the sequences of introns 1 and 2 were determined from 30 different cell lines, encompassing 15 different DQA1 alleles. The sequences revealed major nucleotide differences between the different lineages, whereas within each lineage few differences were present. Phylogenetic analysis of intron and exon sequences confirmed this lineage specificity. Altogether, the present data indicate that the HLA-DQA1 lineages represent ancient entities. The observed variation of the introns in alleles with identical exon sequences implicates conservative selection of the exons within a given lineage. Intron sequences may provide the means to set up an accurate typing system.

Sequence-based typing of exons 1-5 of a new HLA-B allele, B*3927*.

Anew human leucocyte antigen-B (HLA-B) allele, B*3927, was detected in three individuals of a Caucasian family by routine typing with sequence-specific primers (SSP). Serological typing showed B27 Bw4 and B39 Bw6, whereas SSP detected only B*27 as well as the Bw4 and Bw6 motif. The sequence of exons 1-5 of the new allele was determined by allele-specific amplification and sequencing. The new B*39 allele showed one nucleotide difference with B*390101 at position 299 in exon 2. Codon 100 changed from GAG to GTG, resulting in an amino acid substitution from glutamic acid to valine at position 76 of the mature protein. The haplotype carrying the B*3927 allele was A*010101, B*3927, Cw*120301, DRB1*0101 and DQB1*050101.

Identification of a new HLA-C allele, Cw*0316, by sequence-based typing.

The presence of a new allele, Cw*0316, was detected in a Caucasian individual through an unusual association. Molecular typing of the individual by sequence-specific primers and sequence-specific oligonucleotides showed the presence of B*58, B*41 and Cw*17. Sequence-based typing revealed the additional presence of another human leucocyte antigen-C allele. The new allele showed four nucleotide differences with Cw*030202 at positions 559, 560, 589 and 594 in exon 3, leading to three codon changes, codons 187, 197 and 198. This resulted in two amino acid substitutions at positions 163 (L-T) and 173 (K-E) of the mature protein, which proved sufficient to abrogate serological reactivity with Cw3-specific sera.

Ambiguities of human leukocyte antigen-B resolved by sequence-based typing of exons 1, 4, and 5.

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.

Polymorphism of intron 4 in HLA-A, -B and -C genes.

The sequence database of HLA class I genes focuses on the coding sequences, the exons. Limited information is available on the non-coding sequences of the different class I alleles. In this study we have determined the intron 4 nucleotide sequence of at least one representative of each major allelic group of HLA-A, -B and -C. The intron 4 sequences were determined for 27 HLA-A, 81 HLA-B and 30 HLA-C alleles by allele-specific sequencing, using primers located in adjacent exons and introns. The sequences revealed that the length of intron 4 varies with a minimum of 93 and a maximum of 124 nucleotides as a result of insertions and deletions. There were remarkable similarities and differences within HLA-A, -B and -C, as well as between them. Within HLA-A, a deletion of three nucleotides was detected in several HLA-A alleles. The HLA-B alleles could be divided into two groups with one group having a deletion of 11 nucleotides compared with the second group. Within HLA-C, all Cw*07 alleles showed remarkable differences with the other Cw alleles. Cw*07 had an insertion of three nucleotides, shared only by the Cw*17 group. Moreover, Cw*07 was found to have an aberrant nucleotide sequence. Differences between HLA-A, -B and -C alleles were also observed. Remarkable was the deletion of 20 nucleotides in all HLA-A and -B alleles compared with HLA-C, whereas the HLA-A alleles showed an insertion of one nucleotide and a deletion of three nucleotides compared with HLA-B and -C. Furthermore, 32 different polymorphic positions were detected between HLA-A, -B and -C.

Identification of three new DRB1 alleles, DRB1*0107, *0425 and *13012 and confirmation of DRB4*01033.

The characterization of three novel DRB1 alleles is described, DRB1*0107, DRB1*0425 and DRB1*13012 as well as confirmation of DRB4*01033. Two alleles, DRB1*0107 and *0425, showed amino acid differences with previously identified HLA molecules. In DRB1*0107, the glutamine at position 10 was substituted by a glutamic acid. DRB1*0425 showed one amino acid difference with DRB1*0418 (I to F) at position 67, and five amino acid differences with DRB1*04011 at positions 67 (L to F), 70 (Q to D), 71 (K to R), 74 (A to L) and 86 (G to V). The alleles DRB1*13012 and DRB4*01033 had protein sequences identical to DRB1*13011 and DRB4*01031/01032, respectively. Nucleotide differences were present at position 306 for DRB1*13012 and at position 321 for DRB4*01033.

Identification of a new HLA-B*40 variant, B*4035.

In this report, the novel allele B*40351 is presented. The allele was identified in a Caucasian individual by sequence-based typing. B*4035 is identical to B*4002 in exon 2, but differs in exon 3 at position 463, where it has an A in stead of a C. This results in an amino acid change from arginine to serine at codon 131 of the mature protein. The haplotype carrying the B*4035 was A3 B*4035 Cw2 DR11 DQ3.

Molecular and serological identification of the HLA-A*3404 allele.

In this report we describe a novel HLA-A*34 allele, A*3404, which was initially detected by an unusual serological pattern in two unrelated individuals. Sequencing revealed that the new allele was identical to A*3402 in exons 2 and 3, except for a single nucleotide difference at position 238, changing codon 56 from glycine to arginine. The codon change resulted in positive serological reactions with several sera recognizing A30 and/or A31, implicating an important role for this position in epitope recognition. The allele was identified twice on the haplotype A*3404, B*1402, Cw*0802, DRB1*14, DQB1*05.

Sequence analysis of exons 1, 2, 3, 4 and 5 of the HLA-B5/35 cross-reacting group.

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.

B*27 in molecular diagnostics: impact of new alleles and polymorphism outside exons 2 and 3.

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.

Identification of two new HLA-A alleles, A*2419 and A*3011, by sequence-based typing.

In this report, we describe two new HLA-A alleles, A*2419 and A* 3011, that were initially recognized by an aberrant serological pattern. Sequence-based typing revealed sequence differences with other known HLA-A alleles. Allele A*2419 showed 4 nucleotide differences with A*2404, resulting in 4 amino acid differences at codons 70, 76, 77 and 90. Compared with other A*24 alleles, A*2419 lacks the Bw4 motif, as do A*2404 and A*2428. The A*3011 allele showed 2 mismatches with A*3001, resulting in one amino acid difference at codon 80.