Complete HLA genotyping of type 1 diabetes patients and controls from Mali reveals both expected and novel disease associations.

HLA genotyping was performed on 99 type 1 diabetes (T1D) patients and 200 controls from Mali. Next-generation sequencing of the classical HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1, and -DPB1 loci revealed strong T1D association for all loci except HLA-C and -DPA1. Class II association is stronger than class I association, with most observed associations predisposing or protective as expected based on previous studies. For example, HLA-DRB1*03:01, HLA-DRB1*09:01, and HLA-DRB1*04:05 predispose for T1D, whereas HLA-DRB1*15:03 is protective. HLA-DPB1*04:02 (OR = 12.73, p = 2.92 × 10-05 ) and HLA-B*27:05 (OR = 21.36, p = 3.72 × 10-05 ) appear highly predisposing, although previous studies involving multiple populations have reported HLA-DPB1*04:02 as T1D-protective and HLA-B*27:05 as neutral. This result may reflect the linkage disequilibrium between alleles on the extended HLA-A*24:02~HLA-B*27:05~HLA-C*02:02~HLA-DRB1*04:05~HLA-DRB4*01:03~HLA-DQB1*02:02~HLA-DQA1*02:01~HLA-DPB1*04:02~HLA-DPA1*01:03 haplotype in this population rather than an effect of either allele itself. Individual amino acid (AA) analyses are consistent with most T1D association attributable to HLA class II rather than class I in this data set. AA-level analyses reveal previously undescribed differences of the HLA-C locus from the HLA-A and HLA-B loci, with more polymorphic positions, spanning a larger portion of the gene. This may reflect additional mechanisms for HLA-C to influence T1D risk, for example, through expression differences or through its role as the dominant ligand for killer cell immunoglobulin-like receptors (KIR). Comparison of these data to those from larger studies and on other populations may facilitate T1D prediction and help elucidate elusive mechanisms of how HLA contributes to T1D risk and autoimmunity.

Reduced PCR-generated errors from a hybrid capture-based NGS assay for HLA typing.

Next generation sequencing (NGS) assays are state of the art for HLA genotyping. To sequence on an Illumina sequencer, the DNA of interest must be enriched, fragmented, and bookended with known oligonucleotide sequences, a process known as library construction. Many HLA genotyping assays enrich the target loci by long-range PCR (LR-PCR), prior to fragmentation. This PCR step has been reported to introduce errors in the DNA to be sequenced, including inaccurate replication of repeated sequences, and the in vitro recombination of alleles encoded on separate chromosomes. An alternative library construction method involves fragmentation of genomic DNA, followed by hybrid-capture (HC) enrichment of target HLA loci. This HC-based method involves PCR, but with far fewer cycles. Consequently, the HC method had significantly fewer PCR-induced errors, including more faithful replication of repeated sequences, and the near elimination of recombinant sequences. These improvements likely produce more accurate NGS sequencing data of HLA loci.

Development of an immunogenicity score for HLA-DQ eplets: A conceptual study.

Eplets are defined as distinct amino acid configurations on the surface of HLA molecules. The aim of this study was to estimate the immunogenicity of HLA-DQ eplets in a cohort of 221 pregnancies with HLA-DQ mismatches. We defined the immunogenicity of an eplet by the frequency of antibody responses against it. Around 90% of all listed DQB1 or DQA1 eplets were at least five times mismatched and thus included for the calculation of their immunogenicity. The DQB1 eplets with the five highest immunogenicity scores were 55PP, 52PR, 52PQ, 85VG and 45EV; 25% of all DQB1 eplets were not reacting. The DQA1 eplets with the five highest immunogenicity scores were 25YS, 47QL, 55RR, 187T and 18S; 17% of all DQA1 eplets were not reacting. The immunogenicity score had a slightly higher area under the curve to predict development of child-specific antibodies than various molecular mismatch scores (eg, eplet mismatch load, amino acid mismatch load). Overlapping eplets were identified as a barrier to unambiguously assign the immunogenicity score based on HLA antibody reaction patterns. In this conceptual study, we explored the immunogenicity of HLA-DQ eplets and created a map of potentially immunogenic regions on HLA-DQ molecules, which requires validation in clinical transplant cohorts.

Toward defining the immunogenicity of HLA epitopes: Impact of HLA class I eplets on antibody formation during pregnancy.

Eplets are functional units of structural epitopes on donor HLA, potentially recognized by complementarity-determining regions of the paratope of the recipients' B-cell receptors or antibodies (Ab). Their individual immunogenicity is poorly described, yet this feature would be of clinical importance for pretransplant risk assessment. The aim of this study was to determine the relative immunogenicity of HLA class I eplets in the pregnancy setting, where mismatched eplets are present on paternal HLA antigens of the unborn child. One hundred fifty-nine predominantly Caucasian mothers giving birth at the University Hospital Basel and their first newborns were HLA-typed at high-resolution by next-generation sequencing (NGS) (NGSgo Workflow and NGSengine from GenDx; sequencing with a Miseq from Illumina) and eplets were determined using HLAMatchmaker. HLA class I specific IgG Ab was assessed in maternal sera drawn immediately after full-term delivery, by OneLambda LABScreen single antigen ibeads. The Ab profile was subsequently evaluated for eplet-associated patterns. All 72 currently Ab-verified HLA class I eplets were examined for their immunogenicity according to the frequency of child-specific HLA Ab (CSA) directed against their structures. Four hundred twelve of 477 (86.4%) paternal HLA-A, -B or -C alleles were mismatched. CSA were present in 46 mothers (28.9%), directed against 80 (19.4%) of these mismatches. The 10 most immunogenic eplets were 62GK, 145KHA, 144TKH, 62GE, 107W, 80I, 82LR, 41T, 127K, 45KE with immunogenicity rates between 45.8% and 27.3%. This pregnancy study also identified five non-reactive eplets: 62RR, 76ESN, 80TLR, 156DA, 163RW. Based on our results, immunogenic hot and cold spots on the surface of HLA class I molecules were localized and visualized on 3D models. This study strengthens the presumption that different eplets represent different immunogenic potentials. Validation of these results in the clinical transplant setting is an essential next step in identifying those eplets representing a particularly high-risk potential.

The elucidation of KIR2DL4 gene polymorphism.

The killer cell immunoglobulin-like receptors (KIRs) on NK cells recognize defined groups of HLA class I alleles. By this mechanism the NK cells fulfil a significant role in the first line of defense against infectious agents and cancer. For the treatment of leukaemia this NK cell allorecognition is of great importance. Still, an appropriate effect against the leukaemic cells requires sufficient expression of both KIR and HLA proteins. KIR gene polymorphism influence membrane expression of the KIR protein. We addressed KIR2DL4 gene polymorphism by a newly developed DNA and cDNA based direct sequencing based typing (SBT) and cloning approach. A panel of 44 individuals revealed a variety of KIR2DL4 alleles. Three new alleles have been identified, among those one allele showed alternatively spliced products. In conclusion, this approach is applicable for routine KIR2DL4 allele typing and enables the characterisation of new KIR2DL4 alleles.

Activating KIRs exert a crucial role on relapse and overall survival after HLA-identical sibling transplantation.

Recognition of HLA-C molecules by killer cell immunoglobulin-like receptors (KIRs) is an important mechanism in the regulation of natural killer (NK) cell activity. Eradication of residual leukaemic cells by alloreactive donor NK cells after haematopoietic stem cell transplantation (HSCT) fulfils a crucial role in the control of relapse. This retrospective study evaluates 83 patients and their related donors. All individuals were typed at low-resolution level to determine their HLA repertoire. KIR genotyping data were obtained by the use of sequence-specific oligonucleotide (SSO) analysis. All data were combined with patient and donor characteristics and post-transplant clinical data. A higher overall survival was seen when KIR2DS1 in the donor was mismatched with the HLA-C group 2 ligand in the patient (p=0.03). The number of activating KIRs either in the patient or in the donor was significantly correlated with the occurrence of relapse (p=0.003 and p=0.02, respectively). In addition, the presence of KIR2DS5 in the patient alone or in both the patient and donor was significantly correlated with the occurrence of relapse (p=0.004 and p=0.005, respectively). In conclusion, significant correlations were found for activating KIRs with overall survival and relapse.

Patients benefit from the addition of KIR repertoire data to the donor selection procedure for unrelated haematopoietic stem cell transplantation.

Killer cell immunoglobulin-like receptors (KIRs) expressed on donor natural killer (NK) cells are important for induction of NK cell alloreactivity in haematopoietic stem cell transplantation (HSCT). Current criteria in the selection procedure of an unrelated donor do not account for this potential NK alloresponse. In this study the KIR gene repertoire of 21 HSCT patients and all their potential, unrelated donors (N=64) has been identified by the sequence-specific priming (SSP) procedure. KIR genotype characteristics are correlated with HLA and clinical data. These data show that for 16 cases an HLA compatible alternative donor was available. Among those 16 were 8 donors with a favourable predicted NK alloreactivity directed against the leukaemic cells. In conclusion, it is feasible and clinically relevant to add the KIR repertoire to the unrelated donor selection procedure.

Accuracy of NGS HLA typing data influenced by STR.

Next Generation Sequencing (NGS) has become a major technology in HLA typing. The expectations are that highly accurate and unambiguous typing results will be obtained. However, HLA typing by NGS has some limitations caused by imperfections in the PCR amplification. The accuracy of NGS data is investigated by analyzing the Short Tandem Repeats (STR) regions. For this analysis HLA-DRB5 is used as the model. The repeat length in a sample highly influences the repeat length distribution present in the reads of NGS data. With a repeat length of 20 only 50% of all reads were of the estimated repeat length, seriously hampering distinguishing allelic differences in this region correctly. Our findings are confirmed by doing the same analysis in HLA-DRB1. Despite the uncertainty of determining the repeat lengths, several new HLA-DRB5 alleles have been identified in this paper.

Genes in the HLA region indicative for head and neck squamous cell carcinoma.

The majority of genes in the HLA region are directly or indirectly involved in immunological functions. They comprise HLA, HLA-related and non-HLA-related genes. Aberrant HLA expression patterns, including heterogeneous and negative HLA expression, are observed in specimens from head and neck squamous cell carcinoma (HNSCC). To explore the possible role of genes in the HLA region other than the classical HLA genes, susceptibility regions within the HLA region for HNSCC were defined in this study. Microsatellite analysis for 49 microsatellites dispersed throughout the HLA region, in combination with the DNA pooling approach of respectively one control DNA pool and three patient DNA pools, based upon the tumour location, offered an efficient method to define susceptibility regions. In the oral cavity three significant susceptibility regions were localized, one in the class I region (330 kb), and two in the class II region (170 and 210 kb). Eighteen genes from these regions were tested for their RNA expression in oral cavity tumour tissue and compared to expression in the surrounding healthy tissue. A significant increased MICA RNA expression in tumour tissues compared to healthy surrounding tissue and a significant decreased HSD17B8 RNA in tumour tissues compared to surrounding healthy tissue, particular in those tumours without lymph node metastasis, were observed. A trend for decreased RXRbeta and NOTCH4 RNA expression was observed in tumour tissue. In addition to the classical HLA genes, other genes within the HLA region define susceptibility for oral squamous cell carcinoma of the head and neck.

HLA and MICA associations with head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive tumour arising from the epithelial lining of the upper aerodigestive tract. The precise mechanisms involved in the pathogenesis of HNSCC have not been elucidated. Previous studies observed aberrant HLA expression patterns on HNSCC tumour cells and this study focused on the allelic polymorphism of HLA genes and the MHC class I chain related gene A (MICA) and HNSCC. We investigated whether associations with HLA and/or MIC alleles or haplotypes are involved in the pathogenesis of HNSCC and could explain the observed HLA expression patterns. Patients and controls were typed for HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 with sequence specific priming (SSP), supplemented with sequencing based typing (SBT). MICA allelic polymorphism was included and MICA allele assignment was based upon the combination of high resolution SBT of exons 2-4 in combination with repeat analysis and nucleotide polymorphism of exon 5. HLA-B *35 (p=0.014, OR=0.31) and HLA-B *40 (p=0.013, OR=2.9) were significantly associated in respectively the metastasized patients and the oral cavity patients. In addition, the HLA-B *40-DRB1 *13 haplotype (p=0.016, OR=4.1) was more often observed in the oral cavity patient group. The biological significance of the prevalence of specific HLA haplotypes in patients with oral cavity HNSCC and metastasizing HNSCC requires further investigation.

MHC class I chain-related gene a diversity in head and neck squamous cell carcinoma.

Many immune-related genes are located within the human leukocyte antigen (HLA) region on chromosome 6. The MHC class I chain-related gene A (MICA), located centromeric of HLA-B, is involved in the innate and adaptive immune response through activation of NK and T cells. Differences of MICA transmembrane repeat lengths have been associated with diseases and expression is observed on epithelial tumors. Head and neck squamous cell carcinoma (HNSCC) is an epithelial tumor. In the present study we evaluated the MICA repeat length diversity in relation to MICA expression in Dutch HNSCC patients. MICA short tandem repeat analysis indicated a significant decrease in the frequency for the MICA-A9 repeat in patients diagnosed with oral cavity squamous cell carcinoma (SCC) but not in patients with SCC in the hypoharynx, larynx, or oropharynx. Interestingly, the majority of patients expressed MICA as observed with immunohistochemical staining whereas no soluble MICA was detected in patients' sera by enzyme-linked immunosorbent assay. In conclusion, the length of the MICA transmembrane repeats in Dutch HNSCC patients does not influence the MICA expression on tumor cells.

Identification of HLA-A*0111N: a synonymous substitution, introducing an alternative splice site in exon 3, silenced the expression of an HLA-A allele.

A new variant of the HLA-A*010101 allele designated as HLA-A*0111N, previously known as HLA-A*010101var, was identified in a patient requiring a stem-cell transplantation. The patient was typed by serologic methods as HLA-A2 homozygous and by sequence-based typing (SBT) as A*010101,020601. Flow-cytometric (FCM) analysis with 11 human monoclonal antibodies (mAbs) for the A1 molecule confirmed lack of any cell membrane expression of the A*0111N allele. One-dimensional isoelectric focusing (1D-IEF) of total cell lysate from the patient's cells revealed no cell surface and cytoplasmic A1 protein expression, whereas the HLA-A2 molecule was identified by both FCM analysis and 1D-IEF. DNA sequence analysis showed the presence of a synonymous substitution from G to T at position 597 in codon 175. RNA SBT revealed a deletion of 24 bp in exon 3, position 596 through 619, encoding codons 175 through 182 of the HLA-A*0111N allele. The synonymous substitution introduced a new splice site, resulting in an efficient splicing, because no classical A1 protein could be detected in the patient. This alternative splicing prevented the translation into a correct and stable class I molecule expression on the cell surface.

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.

Amino acid residue 67 (isoleucine) of HLA-DRB is associated with POHS.

PURPOSE: To investigate whether presumed ocular histoplasmosis syndrome (POHS) in The Netherlands is associated with HLA-DR2 and HLA-B7, as previously shown in the United States. METHODS: Twenty-four Dutch patients with POHS were included in this study. DNA isolated from peripheral blood leukocytes was typed for HLA by a sequence-based method. Associations were statistically determined. The frequencies of HLA alleles in bone marrow of donors listed on the European donor registry was used to represent the distribution in the normal population. Patients were included in the study only when no cells were present in the vitreous at any time and when fundus photographs fit the diagnosis made according to the following criteria: presence of peripapillary atrophy, presence of punched out chorioretinal lesions (histospots), and presence of a submacular scar. After the fundus photographs were judged, the patients were divided into two groups. Group1 contained patients who met all three diagnostic criteria (complete POHS), and group 2 contained patients who met one or two of the criteria (incomplete POHS). RESULTS: Group 1 consisted of 14 patients and group 2 of 10 patients. An association between POHS and HLA-DR2 and -B7 was present, compared with the normal Dutch control subjects. Although significant, the association between the frequency of HLA-DR2 and -B7 of all patients with POHS was less striking than the findings in patients with POHS in the United States. The association with DR2 in patients with incomplete POHS (group 2) was significantly different from that in the group with complete POHS (group 1). According to the defined criteria the association of POHS with HLA-B7 and -DR2 was confined to the incomplete POHS group and was not found in the complete POHS group. Furthermore, analysis of DR at the amino acid level, rather than at the allele level (DR2) showed that amino acid 67 of the DRB1 alleles had the most significant HLA association with POHS, independent of the two groups. CONCLUSIONS: POHS in Dutch patients was associated with HLA-B7 and -DR2, but more striking was the presence of isoleucine at position 67 of the HLA-DR molecule.

MICA association with presumed ocular histoplasmosis syndrome (POHS).

PURPOSE: MHC class I chain related gene A (MICA), a polymorphic and stress-inducible cell surface molecule, is located centromeric to human leukocyte antigen locus B (HLA-B) in the human leukocyte antigen (HLA) region on chromosome 6. MICA is thought to be involved in the innate immune response. An alanine repeat polymorphism is present in the MICA transmembrane region, for which several disease associations have been reported. Previous research indicated an association with the HLA-B7-DR2 haplotype. In this study we investigated the association of the polymorphic MICA alanine repeat and the ocular disease presumed ocular histoplasmosis syndrome (POHS). METHODS: Twenty-four patients and 106 controls were evaluated for the alanine repeat. A PCR reaction was performed to amplify the polymorphic MICA alanine repeat. Allele lengths of the MICA alanine repeat in patients and controls were determined with GeneScan analysis. RESULTS: No significant associations were observed. Phenotype frequencies of the polymorphic MICA alanine repeat were not significantly different between POHS patients and controls. Neither in the complete patient group compared with the control group nor in one of the subdivided patient groups compared with the control group. CONCLUSIONS: We conclude that the MICA alanine repeat is not a disease-associated factor in POHS. Further analysis of other genes in the B-DR region might elucidate the association of POHS with B7-DR2.

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.