S. Wenda

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 distribution of MICA alleles in an Austrian population: evidence for increasing polymorphism.

The Major Histocompatibility Complex Class I Chain-Related Gene A (MICA) is located 46.4 Kb centromeric to HLA-B locus on chromosome 6; 84 alleles have been described so far. To assess the distribution of MICA alleles in an Austrian population, 322 unrelated Austrian blood donors have been typed for MICA by direct sequencing of amplified exons 2-5; sequencing of exon 6 and separating alleles by haplotype specific primers or by cloning was performed to resolve ambiguities. HLA-B was typed at low level resolution and linkage disequilibrium was determined. We observed 20 already known and four novel MICA alleles. MICA*008:01/04 was the most frequent allele (42%), followed by MICA*002:01 (11%) and MICA*009:01 (9%), three alleles (MICA*029, *067 and *068) were observed only once. No deviation from the Hardy Weinberg equilibrium was observed. Linkage disequilibrium between MICA and HLA-B alleles was observed, most extensively between MICA*008:01/04 and HLA-B*07. Our population data are in agreement with other European populations. The fact that four novel alleles have been observed indicates that the polymorphism of MICA is larger than currently estimated.

Administration of recombinant human granulocyte-colony-stimulating factor does not induce long-lasting detectable epigenetic alterations in healthy donors.

The short‐term safety profile of recombinant human granulocyte–colony‐stimulating factor (rHuG‐CSF) in the allogeneic stem cell setting seems acceptable; only few data on long‐term safety are available. To further study possible epigenetic alterations, we investigated prospectively the influence of rHuG‐CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes in peripheral blood cells of healthy unrelated stem cell donors within an observation period of 1 year.

Sequence-based definition of eight short tandem repeat loci located within the HLA-region in an Austrian population

Sequenced allelic ladders are a prerequisite for reliable genotyping of short tandem repeat (STR) polymorphisms and consistent results across instrument platforms. For eight STR-loci located on the short arm of chromosome 6 (6p21.3), a sequenced based nomenclature was established according to international recommendations. Publicly available reference DNA samples were sequenced enabling interested laboratories to construct their own allelic ladders. Three tetrameric (D6S2691, D6S2678, DQIV), one trimeric (D6S2906) and four dimeric repeat loci (D6S2972, D6S2792, D6S2789, D6S273) were investigated. Apart from the very complex sequence structure at the DQIV locus, three loci showed a compound and four loci a simple repeat pattern. In the flanking regions of some loci additional single nucleotide and insertion/deletion polymorphisms occurred as well as sequence polymorphisms within the repeat region of alleles with the same length. In an Austrian Caucasoid population sample (n=293) between eight and 22 alleles were found. No significant deviation from Hardy-Weinberg expectations was observed, the power of discrimination ranged from 0.826 to 0.978. The loci cover the HLA-coding region from HLA-A to HLA-DQB1 and can be used for a better definition of HLA haplotypes for population and disease association studies, recombination point mapping, haematopoietic stem cell transplantation as well as for identity and relationship testing.

An HLA-B7-specific antibody in an HLA-B*07 positive patient explained by a nonexpressed allele (HLA-B*07:181N)

Antibody identification by a bead array assay in a kidney patient revealed several HLA‐specific antibodies including one directed against the HLA‐B7 antigen. Low‐resolution typing of the patient indicated the presence of an HLA‐B*07 allele. To rule out an HLA‐specific autoantibody the HLA‐typing of the patient was further refined by nucleotide sequencing on a next‐generation sequencing platform and eventually showed an HLA‐B*39:01:01:03 and HLA‐B*07:181N genotype. Thereby the allospecific nature of the antibody was proven. The HLA‐B7‐specific antibody could be explained by an immunization during the first kidney‐transplantation in 1996 with an HLA‐B*07 positive donor. When assessing the plausibility of antibodies, the presence of nonexpressed alleles should be taken into consideration.

Standardized genotyping of HLA STR by CE as surrogate for HLA class I and II markers and for identification of HLA identical siblings.

Linkage disequilibria (LD) between alleles and haplotypes of human leucocyte antigen, locus A (HLA) and STR loci located in the human major histocompatibility complex were analyzed in order to investigate whether or not HLA alleles and haplotypes are predictable by alleles or haplotypes of HLA STRs. Standardized genotyping of eight STR loci (D6S2972, D6S2906, D6S2691, D6S2678, D6S2792, D6S2789, D6S273, and DQIV) was performed by CE on 600 individuals from 150 Austrian Caucasoid families with known HLA‐A,‐B,‐C and –DRB1 typing. From those, 576 full haplotypes of four HLA and eight STR loci were obtained. Haplotypes of two flanking STRs predicted HLA alleles and two‐locus HLA haplotypes better than single STR alleles, except HLA‐DRB1 alleles (92% were in LD with DQIV alleles only). A percentage of 65–86% of three and four‐locus HLA haplotypes were in LD with haplotypes of three, four, and eight of their flanking STR loci including numerous clear‐cut predictions (20–61%). All eight and a set of the four most informative STR loci D6S2972, D6S2678, D6S2792, and DQIV could identify all HLA identical and nonidentical siblings in 138 pairs of siblings. The results of this proof of concept study in Austrian Caucasoids show, that HLA STRs can aid the definition of HLA‐A,‐B,‐C,‐DRB1 haplotypes and the selection of sibling donors for stem cell transplantation.