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The current practice of HLA genotyping in deceased donors poses challenges due to limited resolution within time constraints. Nevertheless, the assessment of compatibility between anti-HLA sensitized recipients and mismatched donors remains a critical medical need, particularly when dealing with allele-specific (second field genotyping level) donor-specific antibodies. In this study, we present a customized protocol based on the NanoTYPE® HLA typing kit, employing the MinION® sequencer, which enables rapid HLA typing of deceased donors within a short timeframe of 3.75 h on average at a three-field resolution with almost no residual ambiguities. Through a prospective real-time analysis of HLA typing in 18 donors, we demonstrated the efficacy and precision of our nanopore-based method in comparison to the conventional approach and without delaying organ allocation. Indeed, this duration was consistent with the deceased donor organ donation procedure leading to organ allocation via the French Biomedicine Agency. The improved resolution achieved with our protocol enhances the security of organ allocation, particularly benefiting highly sensitized recipients who often present intricate HLA antibody profiles. By overcoming technical challenges and providing comprehensive genotyping data, this approach holds the potential to significantly impact deceased donor HLA genotyping, thereby facilitating optimal organ allocation strategies., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Associations between HLA genotype and disease susceptibility encompass almost all the classic HLA loci. The level of typing resolution enabling a correct identification of an HLA disease susceptibility gene depends on the disease itself and/or on the accumulated knowledge about the molecular involvement of the HLA allele(s) engaged. Therefore, the application of Next Generation Sequencing technologies to HLA disease association, which would improve typing resolution, could prove useful to better understand disease severity. In the present study, we tested a nanopore sequencing approach developed by Omixon Biocomputing Ltd, dedicated to on-demand locus typing for HLA and disease, as an alternative to the conventional widely used sequence specific oligoprobe (SSO) approach. A total of 145 DNA samples used in routine diagnosis by SSO were retrospectively analyzed with nanopore technology, for HLA-A*02 immunotherapy decision for A*29, B*27, B*51, B*57 identification in class I, and DRB1, DQA1, and DQB1 for bullous dermatosis, rheumatoid arthritis, diabetes, and celiac disease requests in class II. Each locus was typed in a separate experiment, except for DQB1 and DQA1, which were analyzed together. Concordance between typings reached 100% for all the loci tested. Ambiguities by nanopore were only found for missing exon coverage. This approach was found to be very well adapted to the routine flow imposed by the SSO technique. This study illustrates the use of the new NanoTYPE MONO kit for single locus HLA sequencing for HLA and disease association diagnosis., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Several technical limitations of Luminex single antigen (LSA) assays have been described so far. This study focused on a reactivity pattern observed in many sera that cannot be explained by eplets described in the Epitope Registry database and sometimes appearing against a self-HLA allele or antigen. In most cases, this pattern is revealed by a discrepant result when compared with other assays (Luminex PRA, cell-binding assays such as flow cytometry cross match, LSA from another manufacturer…). We focus here on the Cw1/12/15 pattern appearing on the LABScreen class I LSA provided by One Lambda. We documented its behavior using this LSA after acid denaturation of the beads, using Lifecodes LSA from Immucor, and adsorption of sera either on spleen mononuclear cells from deceased donors or on single HLA transfected cell clones. We studied 33 sera from different patients positive for the three Cw beads, selected from our routine patients' LSA database. Nine patients had transplants from a Cw12 or Cw15 donor without any pejorative evolution of the graft, nor post-transplant MFI (mean fluorescence intensity) increase of the Cw1/12/15 beads. A significant increase of MFI was observed after acid denaturation of the LABScreen beads. All sera tested by Lifecodes LSA were negative for these Cw beads. Finally, we found no significant difference of MFI after adsorption on cells from either origin. Therefore, the Cw1/12/15 pattern appears to be a false positive reactivity of the LABScreen single antigen assay., (© 2023 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

HLA-DQA1*01:81 differs from HLA-DQA1*01:02:01:01 by one nucleotide substitution in codon 153 in exon 3., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Complement-mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti-HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre-treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE-conjugated secondary Ab (IgGPE), an Alexa Fluor 532-conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE-conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Luminex single antigen flow beads (SAFB) assays are used to monitor anti-HLA antibodies, which are associated with allograft loss. The primary antibody binding level is evaluated by the mean fluorescence intensity (MFI) provided by the tracing secondary antibody. The aim of this study was to compare the MFI obtained with two secondary antibodies and to evaluate their sensitivity to detect newly developed de novo anti-HLA donor-specific antibodies (dnDSA). A total of 23 and 46 different sera from 20 HLA-sensitized kidney transplant recipients were tested with class I and II SAFB, respectively, as well as sera from 17 patients with dnDSA. The conventional secondary antibody (IgHPolyFab) was the phycoerythrin (PE)-conjugated goat anti-human IgG constant heavy chain (HC)-binding polyclonal-F(ab')2. The alternate secondary antibody was a PE-conjugated anti-human IgG Fc-specific monoclonal IgG (FcMonoIgG). MFI of negative control sera were drastically lower with the FcMonoIgG than with IgHPolyFab, requesting a unique positivity threshold to be defined for each secondary antibody. MFI obtained with positive control sera were also lower with FcMonoIgG. In kidney transplant recipient's sera, the number of antigenic specificities detected by each secondary antibody was comparable, for dnDSA as well as for nonDSA. In conclusion, decision thresholds must be refined when changing the secondary tracing reagent in SAFB assays. FcMonoIgG did not demonstrate any superiority over IgHPolyFab for monitoring HLA antibodies., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Recent advances in technologies such as high density array-based genotyping, DNA and RNA deep sequencing, expression quantitative-trait loci mapping, epigenome analyses, and new computational strategies, have introduced drastic shifts into our understanding of major histocompatibility complex (MHC) association with immune-mediated diseases. Here, we review the most exciting findings in this field. We show how the fine characterization of structural features and expression levels of MHC alleles, posttranslational or environmental modifications of HLA-bound peptides, and epistatic interactions with non-HLA genes, has made it possible not only to provide mechanistic explanations for MHC associations with immune-mediated diseases but also to help choose relevant therapeutic targets., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The association of human leukocyte antigen (HLA) molecules with many autoimmune diseases has been long known. Yet, the molecular basis for these associations remains unclear for most of these diseases because of the lack of identification of a primary target autoantigen or autoantigens. In two frequent autoimmune disorders, however, celiac disease and type 1 diabetes, recent progress in the identification of immunogenic antigen epitopes and analysis of crystal structure of particular HLA molecules in complex with disease-specific epitopes has allowed for a better understanding of the molecular mechanisms underlying disease association. In this review, these two diseases will be analyzed in detail to show how HLA polymorphisms may directly contribute to susceptibility to, or protection from, disease. Such analyses have significant interest in clinical practice to identify at-risk individuals and elaborate new therapeutic strategies aiming at inhibiting or preventing the autoimmune process.

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.

In Moroccan patients, predisposition to Behçet's disease is associated with HLA-B*51, mostly in males with young age at disease onset. In addition, the disease is associated with B*15 both in females and in males with late disease onset. We analyzed the clinical presentation, the severity and the course of the disease in 86 Moroccan patients according to their HLA-B phenotype. The presence of the B*51 or B*15 did not predispose to a particular clinical manifestation, nor to a more severe presentation of the disease. By contrast, outcome of the disease significantly differed depending on HLA-B phenotype, with an increase of symptoms in most B*51+ patients and in half of B*15 patients, and a remission or a decrease of symptoms in all B*51-B*15- patients. This variable course was mostly observed for ocular lesions, skin lesions, articular symptoms, and neurological symptoms. These data may suggest that treatment should be given early in the course of the disease in B*51 or B*15-positive patients in order to stabilize the inflammatory process.

Coeliac disease (CD) is a multifactorial disease for which there is an intensive search for genetic risk factors. Some authors found an association between the CTLA-4 region and CD. In the present work, we investigate the possible implication of the CTLA-4 region as a genetic risk factor for CD, through two statistical approaches: the maximum likelihood score (MLS) test in a large Italian sample of affected sib-pairs using polymorphic genetic markers on chromosome 2, and the transmission disequilibrium test (TDT) in continental Italian and Tunisian families using the CTLA-4 exon 1 49 A/G polymorphism. None of these approaches provides evidence for linkage or association between the CTLA-4 region and CD. This might result from a difference in the CTLA-4 region from population to population, either in its involvement as a risk factor or in the strength of linkage disequilibrium.

HLA DRB1, DQA1 and DQB1 polymorphisms were analyzed by PCR-SSO typing in a sample of the Moroccan population from Souss. Uneven allelic frequency distributions are observed at each locus, with particularly high frequencies for DRB1*0701, DRB1*0301, DQA1*0501, DQA1*0201, and DQB1*0201. Only three haplotypes (DRB1*0701-DQA1*0201-DQB1*0201, DRB1*0301-DQA1*0501-DQB1*0201 and DRB1*11-DQA1*0501-DQB1*0301) account for nearly 50% of the total gene frequencies. A genetic distance analysis reveals that the Moroccan population is close to the Spanish and the Algerians, who live in geographically neighboring areas. However, the Souss population is also characterized by a lower level of genetic diversity compared to other African and European populations from the Mediterranean area. This may be the result of a rapid genetic drift due to their likely geographical and/or cultural isolation.

TAP, LMP and DM genes map within the major histocompatibility complex (MHC) class II region between the DQB1 and DPB1 loci, and are involved in the processing of peptides bound to HLA class I or class II molecules. In order to determine the various linkage disequilibria existing between these genes and HLA class II genes, we have analyzed TAP1, TAP2, LMP2, DMA, DMB, DRB1, DQA1, DQB1 and DPB1 polymorphisms in 162 unrelated healthy Caucasian individuals. Many positive or negative associations were observed between alleles at these loci, such as between DR/DQ and TAP2, DM or LMP, between DP and DMB, and between TAP2 and DM, TAP2 and LMP. Conversely, no linkage disequilibrium was detected between some closely related genes (DR/DQ and TAP1, TAP1 and TAP2, LMP2 and DM), in agreement with the existence of recombination hot spots in this region. Other weak linkage disequilibria are likely to exist in this region. These data allow to define some conserved MHC class II haplotypes including HLA class II and TAP, LMP and DM alleles. Furthermore, the knowledge of such linkage disequilibria is of outstanding importance in order to avoid misinterpretation of the data when studying MHC class II associations with autoimmune diseases.