Erik H. Rozemuller

High-resolution HLA-DPB typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2.

To differentiate 32 HLA-DPB alleles, conventional techniques such as serology and cellular typing are inadequate for high-resolution DPB typing. The most refined DNA typing until now is SSO typing and new selected oligonucleotides can be added to this system to distinguish new allele sequences. DNA sequencing, however, reveals directly the sequence information of all polymorphic HVRs and has the advantage of being independent from exon polymorphisms. We have developed a new DNA-based typing approach that is rapid, fully automated, and therefore suitable for routine typing. The system is based upon direct sequencing of amplified DNA with fluorescent-labeled primers. The designation of alleles is obtained by a comparison of all polymorphic positions in the determined sequence with all known allele sequences retained in a database along with their heterozygous combinations. Sequence data at both constant and polymorphic positions are used for quality control. In this study, the typing results of a panel of 91 previous SSO-typed DNA samples are described. After comparison with the SSO-typing results, we conclude that with this SBT system allele assignment is reliable. The method is easy to perform since both sequencing and assignment are automated. Furthermore, the system is easily applicable to other gene systems.

Assignment of HLA-DPB alleles by computerized matching based upon sequence data

Routine HLA typing by serology has been supported by DNA or protein analysis of the respective molecules in cases when serologic typing was inconclusive or difficult to perform. DNA analysis by RFLP, SSO, or PCR amplification with SSP is a reliable tool for the identification of alleles. Because DNA sequences may now be determined routinely, we developed software based on sequence data from known sequences for allele assignment of polymorphic gene systems. We describe the assignment of HLA-DPB alleles based upon sequence data obtained after PCR amplification and subsequent sequencing of exon 2. Our software includes a database containing all known HLA-DPB sequences, which offers the possibility of analyzing heterozygous individuals by combinatorial comparison through all alleles and thus identifying the one or two alleles involved. Quality control of the sequence has been included by the alignment of constant regions of the sequence combined with related polymorphism of known polymorphic nucleotides and the identification of the positions crucial for the allele assignment. The ability to type for HLA-DPB alleles routinely by automatic sequence determination and subsequent automated analysis offers new perspectives for HLA-DNA typing.

Mutation in the β2m gene is not a frequent event in head and neck squamous cell carcinomas

Abstract In cryostat sections of 84 head and neck squamous cell carcinomas (HNSCC) HLA class I and β2m expression was analysed using monomorphic and locus specific monoclonal antibodies. Loss of expression was heterogeneous and none of the tumours tested showed a total loss of HLA class I and/or β2m when analysed with W6/32, which recognises HLA class I determinants and anti-β2m MoAbs. Weak HLA class I and β2m expression was found in 9 tumours (11%) and heterogeneous expression was found in 2 tumours (2%). When analysed with locus-specific antibodies (HCA2 and HC10, anti-HLA-A and anti-HLA-B/C, respectively) 37 tumours (44%) showed a loss, weak or heterogeneous expression of one or both loci. Tumours showing a down-regulated HLA class I expression were analysed for mutations in either allele of the β2m gene by sequencing based mutation analysis (SBMA). Exon 1 and exons 2 and 3 were amplified separately by PCR using M13-tailed intron-specific primers. PCR products were sequenced in two directions. In none of the tumours mutations in the β2m gene were detected. In 59% of the tumours with down-regulated HLA class I expression, lost or down-regulated TAP 1 expression was found when analysed with anti-TAP 1 antibodies. This indicates an important role for TAP in down-regulation of HLA class I expression in HNSCC.

Characterization and distribution of Mhc-DPB1 alleles in chimpanzee and rhesus macaque populations

Allelic diversity at the nonhuman primate Mhc-DPB1 locus was studied by determining exon 2 nucleotide sequences. This resulted in the detection of 17 chimpanzee (Pan troglodytes), 2 orangutan (Pongo pygmaeus) and 16 rhesus macaque (Macaca mulatta) alleles. These were compiled with primate Mhc-DPB1 nucleotide sequences that were published previously. Based upon the results, a sequence specific oligotyping method was developed allowing us to investigate the distribution of Mhc-DPB1 alleles in distinct chimpanzee and rhesus macaque colonies. Like found in humans, chimpanzee and rhesus macaque populations originating from different geographic backgrounds appear to be characterized by the presence of a few dominant Mhc-DPB1 alleles.

Donor interleukin-4 promoter gene polymorphism influences allograft rejection after heart transplantation.

BACKGROUND The cytokine interleukin-4 (IL-4) is secreted mainly by activated T lymphocytes and characterizes the T-helper 2 (Th2) sub-type. In transplantation Th2 cells are believed to induce graft tolerance. Previous studies revealed that patients with a relatively high frequency of IL-4 producing helper T lymphocytes (HTL) before heart transplantation (HTX) had no or less rejection episodes compared with patients with a low frequency of IL-4 producing HTL. Three single nucleotide polymorphisms (SNPs) have been identified in the promoter region of the IL-4 gene, which influence promoter strength. We investigated whether there was a correlation between SNP genotypes in the IL-4 promoter and heart failure, and rejection after HTX. METHODS Seventy HTX patients, 61 donors, and 36 controls were genotyped for the 3 SNPs by sequencing. RESULTS Of the SNPs at -285 and -81, only the C and A alleles, respectively, were found in this study. Both alleles were found for the -590 SNP. No relation between patient genotype of the SNP at -590 and heart failure and rejection was found. However, incidence of rejection was significantly lower in patients that received a donor heart with the T-positive genotype compared with patients that received a heart from a T-negative donor. Patients who had the T-negative genotype and received a heart from a T-positive donor, suffered significantly less from rejection than T-negative patients that received a T-negative donor heart. This was not significant in the T-positive patient group. CONCLUSIONS This indicates that IL-4 production within the donor heart and by cells from the donor is important for reducing incidence of episodes of rejection.

Extended HLA-DPB1 polymorphism: an RNA approach for HLA-DPB1 typing

Most of the 119 human leukocyte antigen (HLA)-DPB1 alleles are defined by polymorphism in six hypervariable regions (HVRs) in exon 2 of the HLA-DPB1 gene. We investigated how DPB1 polymorphism is represented in the entire coding region. An RNA sequencing-based typing (SBT) approach was developed for the identification of HLA-DPB1 polymorphism from the 5′ untranslated region (UTR) through the 3′-UTR. B-cell lymphoblastoid cell lines, encoding 16 different DPB1 alleles, were studied. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5′ and 3′-UTR. Four new HLA-DPB1 alleles were identified, DPB1*0502, DPB1*0602, DPB1*0802 and DPB1*0902, which have exon 2 sequences identical to other DPB1 alleles but differ in the extended region. The additional polymorphism represents two main polymorphic lineages in the DPB1 alleles. Among the HVRs in exon 2, only HVR F correlates with these two main lineages.

A COMPUTERIZED METHOD TO PREDICT THE DISCRIMINATORY PROPERTIES FOR CLASS II SEQUENCING BASED TYPING

Accurate identification of alleles or groups of alleles is obtained using DNA techniques for HLA class II typing. In PCR-SSP and PCR-SSO the typing resolution is dependent on the location of the amplification primers and the number of oligonucleotide probes used for hybridization. These primers are developed based upon the known allele sequences. However, the number of published allele sequences increases rapidly. Periodic reconsideration of the discriminatory properties of the methods used is necessary and often leads to the addition of primers, and an increase of ambiguous genotypes. SBT is a high-resolution class II typing method which utilizes the second exon sequence at all positions for typing. Using a generic amplification and sequencing approach, a limited number of ambiguous heterozygotes can be expected. However, the number of ambiguities is dependent on the location of the primers. These ambiguities can be solved by the use of group-specific primers in either the amplification or sequencing reaction. To identify the applicability of a SBT approach with respect to typing resolution, it is important to identify these ambiguities and develop strategies to solve them. We developed software that predicts the ambiguous heterozygotes, and analyzes their sequences for group specific primers, solving these ambiguities. The location of the primers is considered as well as the use of generic vs group-specific amplification or sequencing reactions. Allele sequences of all class II genes, as accepted by the WHO nomenclature committee for factors of the HLA system and submitted to the EMBL/Genbank databases, are analyzed to define the allelic discrimination level of HLA-DQB1, -DQA1, -DFB1, -DPA1, -DRB1, -DRB3, -DRB4, and -DRB5.

No association between transforming growth factor β gene polymorphism and acute allograft rejection after cardiac transplantation

Abstract Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, which inhibits both development of Th1 and Th2 subsets and the Th1 proinflammatory response. TGF-β1 production is influenced through several single nucleotide polymorphisms (SNP) in the structural gene and promoter region. Acute rejection of transplants depends on the Thl/Th2 balance within the graft, high levels of TGF-β1 shift this balance towards Th2. We investigated whether genotypes of 4 SNP (−800 and −509 in the promoter region, codon 10 and codon 25 in the first exon) were correlated with cardiac disease or with incidence of rejection after heart transplantation (HTX). Genotypes were determined for 70 HTX patients and 61 donors by sequencing or oligonucleotide ligation assay. No association between SNP genotypes and heart disease or acute transplant rejection was observed. We conclude that genetic variation in the TGF-β1 gene neither influences the existence of cardiomyopathy nor the incidence of rejection upon HTX.

Role of Human Leucocyte Antigen DQ in the Presentation of T Cell Epitopes in the Major Cow’s Milk Allergen αs1-Casein

Background: Little is known about the association between human leucocyte antigen (HLA) and cow’s milk allergy (CMA). The aim of the present study was to determine the HLA restriction of T cell clones (TCCs) specific to αs1-casein, the most abundant milk protein, and to study possible HLA class II allele associations with CMA. Methods: αs1-Casein-specific TCCs were derived from 6 children with CMA, 9 atopic children without CMA and 5 non-atopic children. T cell epitope specificity was defined by stimulation with overlapping peptides, spanning the αs1-casein molecule. HLA restriction was determined in proliferation assays using antibodies blocking either HLA-DP, HLA-DQ or HLA-DR. HLA genotyping was performed in 32 subjects with CMA, 23 atopic and 22 non-atopic individuals. Results: Ten TCCs were restricted to HLA-DQ, 6 TCCs to HLA-DR and 4 TCCs to HLA-DP. The sequence in αs1-casein that was most immunogenic to T cells from children with CMA contained T cell epitopes restricted to DQB1*0201, DPB1*0401 and DRB1*1501. The DQB1*0501 allele frequency was lower in children with CMA than in non-atopic children, but this difference could not be confirmed in an additional group of subjects with and without CMA. Conclusions: HLA-DQ plays a substantial role in the presentation of T cell epitopes in αs1-casein. However, HLA class II allele frequencies do not show major differences between cow’s milk allergic, atopic and non-atopic subjects. T cell epitopes in the most immunogenic region are presented by various abundantly present HLA genotypes. Therefore, this sequence may be a suitable target for peptide immunotherapy.

Minimum information for reporting next generation sequence genotyping (MIRING): Guidelines for reporting HLA and KIR genotyping via next generation sequencing

The development of next-generation sequencing (NGS) technologies for HLA and KIR genotyping is rapidly advancing knowledge of genetic variation of these highly polymorphic loci. NGS genotyping is poised to replace older methods for clinical use, but standard methods for reporting and exchanging these new, high quality genotype data are needed. The Immunogenomic NGS Consortium, a broad collaboration of histocompatibility and immunogenetics clinicians, researchers, instrument manufacturers and software developers, has developed the Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines. MIRING is a checklist that specifies the content of NGS genotyping results as well as a set of messaging guidelines for reporting the results. A MIRING message includes five categories of structured information - message annotation, reference context, full genotype, consensus sequence and novel polymorphism - and references to three categories of accessory information - NGS platform documentation, read processing documentation and primary data. These eight categories of information ensure the long-term portability and broad application of this NGS data for all current histocompatibility and immunogenetics use cases. In addition, MIRING can be extended to allow the reporting of genotype data generated using pre-NGS technologies. Because genotyping results reported using MIRING are easily updated in accordance with reference and nomenclature databases, MIRING represents a bold departure from previous methods of reporting HLA and KIR genotyping results, which have provided static and less-portable data. More information about MIRING can be found online at miring.immunogenomics.org.