HLA*LA-HLA typing from linearly projected graph alignments.

SUMMARY: HLA*LA implements a new graph alignment model for human leukocyte antigen (HLA) type inference, based on the projection of linear alignments onto a variation graph. It enables accurate HLA type inference from whole-genome (99% accuracy) and whole-exome (93% accuracy) Illumina data; from long-read Oxford Nanopore and Pacific Biosciences data (98% accuracy for whole-genome and targeted data) and from genome assemblies. Computational requirements for a typical sample vary between 0.7 and 14 CPU hours per sample. AVAILABILITY AND IMPLEMENTATION: HLA*LA is implemented in C++ and Perl and freely available as a bioconda package or from https://github.com/DiltheyLab/HLA-LA (GPL v3). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Distinguishing functional polymorphism from random variation in the sequences of >10,000 HLA-A, -B and -C alleles.

HLA class I glycoproteins contain the functional sites that bind peptide antigens and engage lymphocyte receptors. Recently, clinical application of sequence-based HLA typing has uncovered an unprecedented number of novel HLA class I alleles. Here we define the nature and extent of the variation in 3,489 HLA-A, 4,356 HLA-B and 3,111 HLA-C alleles. This analysis required development of suites of methods, having general applicability, for comparing and analyzing large numbers of homologous sequences. At least three amino-acid substitutions are present at every position in the polymorphic α1 and α2 domains of HLA-A, -B and -C. A minority of positions have an incidence >1% for the 'second' most frequent nucleotide, comprising 70 positions in HLA-A, 85 in HLA-B and 54 in HLA-C. The majority of these positions have three or four alternative nucleotides. These positions were subject to positive selection and correspond to binding sites for peptides and receptors. Most alleles of HLA class I (>80%) are very rare, often identified in one person or family, and they differ by point mutation from older, more common alleles. These alleles with single nucleotide polymorphisms reflect the germ-line mutation rate. Their frequency predicts the human population harbors 8-9 million HLA class I variants. The common alleles of human populations comprise 42 core alleles, which represent all selected polymorphism, and recombinants that have assorted this polymorphism.

Donor Killer Cell Immunoglobulin-Like Receptor Genotype Does Not Improve Graft-versus-Leukemia Responses in Chronic Lymphocytic Leukemia after Unrelated Donor Transplant: A Center for International Blood and Marrow Transplant Research Analysis.

Allogeneic hematopoietic cell transplantation (alloHCT) remains the sole curative therapy for patients with chronic lymphocytic leukemia (CLL), leading to 40% to 45% long-term survival. The impact of donor killer immunoglobulin-like receptor (KIR) genotype on outcomes of unrelated donor (URD) alloHCT for CLL is unknown. We examined 573 adult URD CLL recipient pairs. KIR genotype (presence/absence) was determined for each donor, and comprehensive modeling of interactions with recipient HLA class I loci (KIR ligands) was used to evaluate their effect on relapse and survival. Recipients had a median age of 56 years, and most were not in remission (65%). Both 8/8 HLA-matched (81%) or 7/8 HLA matched grafts (19%) were studied. Factors associated with improved overall survival (OS) were reduced-intensity conditioning (hazard ratio [HR] of death, .76) and good performance status (HR, .46), whereas alloHCT in nonremission (HR, 1.96) and mismatched donors (HR, 2.01) increased mortality. No models demonstrated a relationship between donor KIR genotype and transplant outcomes. Cox regression models comparing donors with A/A versus B/x KIR haplotypes and those with KIR gene content scores of 0 versus 1 versus ≥2 yielded similar rates of nonrelapse mortality, relapse, acute graft-versus-host disease (GVHD), and chronic GVHD and the same progression-free survival and OS. Relapse risk was not different for grafts from donors with KIR3DL1 transplanted into HLA C1/1 versus C2 recipients. This large analysis failed to demonstrate an association between URD KIR genotype and transplant outcome for patients with CLL, and thus KIR genotyping should not be used as a donor selection criterion in this setting.

High-Accuracy HLA Type Inference from Whole-Genome Sequencing Data Using Population Reference Graphs.

Genetic variation at the Human Leucocyte Antigen (HLA) genes is associated with many autoimmune and infectious disease phenotypes, is an important element of the immunological distinction between self and non-self, and shapes immune epitope repertoires. Determining the allelic state of the HLA genes (HLA typing) as a by-product of standard whole-genome sequencing data would therefore be highly desirable and enable the immunogenetic characterization of samples in currently ongoing population sequencing projects. Extensive hyperpolymorphism and sequence similarity between the HLA genes, however, pose problems for accurate read mapping and make HLA type inference from whole-genome sequencing data a challenging problem. We describe how to address these challenges in a Population Reference Graph (PRG) framework. First, we construct a PRG for 46 (mostly HLA) genes and pseudogenes, their genomic context and their characterized sequence variants, integrating a database of over 10,000 known allele sequences. Second, we present a sequence-to-PRG paired-end read mapping algorithm that enables accurate read mapping for the HLA genes. Third, we infer the most likely pair of underlying alleles at G group resolution from the IMGT/HLA database at each locus, employing a simple likelihood framework. We show that HLA*PRG, our algorithm, outperforms existing methods by a wide margin. We evaluate HLA*PRG on six classical class I and class II HLA genes (HLA-A, -B, -C, -DQA1, -DQB1, -DRB1) and on a set of 14 samples (3 samples with 2 x 100bp, 11 samples with 2 x 250bp Illumina HiSeq data). Of 158 alleles tested, we correctly infer 157 alleles (99.4%). We also identify and re-type two erroneous alleles in the original validation data. We conclude that HLA*PRG for the first time achieves accuracies comparable to gold-standard reference methods from standard whole-genome sequencing data, though high computational demands (currently ~30-250 CPU hours per sample) remain a significant challenge to practical application.

Identification of the novel HLA-B*53:69 allele by sequencing-based typing.

The HLA-B*53:69 allele differs from HLA-B*53:01:01:01 by two nucleotide changes in exon 3.

Exploration of potential immune mechanisms in cervical dystonia.

BACKGROUND: Although there are many possible causes for cervical dystonia (CD), a specific etiology cannot be identified in most cases. Prior studies have suggested a relationship between autoimmune disease and some cases of CD, pointing to possible immunological mechanisms. OBJECTIVE: The goal was to explore the potential role of multiple different immunological mechanisms in CD. METHODS: First, a broad screening test compared neuronal antibodies in controls and CD. Second, unbiased blood plasma proteomics provided a broad screen for potential biologic differences between controls and CD. Third, a multiplex immunoassay compared 37 markers associated with immunological processes in controls and CD. Fourth, relative immune cell frequencies were investigated in blood samples of controls and CD. Finally, sequencing studies investigated the association of HLA DQB1 and DRB1 alleles in controls versus CD. RESULTS: Screens for anti-neuronal antibodies did not reveal any obvious abnormalities. Plasma proteomics pointed towards certain abnormalities of immune mechanisms, and the multiplex assay pointed more specifically towards abnormalities in T lymphocytes. Abnormal immune cell frequencies were identified for some CD cases, and these cases clustered together as a potential subgroup. Studies of HLA alleles indicated a possible association between CD and DRB1*15:03, which is reported to mediate the penetrance of autoimmune disorders. CONCLUSIONS: Altogether, the association of CD with multiple different blood-based immune measures point to abnormalities in cell-mediated immunity that may play a pathogenic role for a subgroup of individuals with CD.

High-resolution African HLA resource uncovers HLA-DRB1 expression effects underlying vaccine response.

How human genetic variation contributes to vaccine effectiveness in infants is unclear, and data are limited on these relationships in populations with African ancestries. We undertook genetic analyses of vaccine antibody responses in infants from Uganda (n = 1391), Burkina Faso (n = 353) and South Africa (n = 755), identifying associations between human leukocyte antigen (HLA) and antibody response for five of eight tested antigens spanning pertussis, diphtheria and hepatitis B vaccines. In addition, through HLA typing 1,702 individuals from 11 populations of African ancestry derived predominantly from the 1000 Genomes Project, we constructed an imputation resource, fine-mapping class II HLA-DR and DQ associations explaining up to 10% of antibody response variance in our infant cohorts. We observed differences in the genetic architecture of pertussis antibody response between the cohorts with African ancestries and an independent cohort with European ancestry, but found no in silico evidence of differences in HLA peptide binding affinity or breadth. Using immune cell expression quantitative trait loci datasets derived from African-ancestry samples from the 1000 Genomes Project, we found evidence of differential HLA-DRB1 expression correlating with inferred protection from pertussis following vaccination. This work suggests that HLA-DRB1 expression may play a role in vaccine response and should be considered alongside peptide selection to improve vaccine design.

Recombinant structures expand and contract inter and intragenic diversification at the KIR locus.

BACKGROUND: The human KIR genes are arranged in at least six major gene-content haplotypes, all of which are combinations of four centromeric and two telomeric motifs. Several less frequent or minor haplotypes also exist, including insertions, deletions, and hybridization of KIR genes derived from the major haplotypes. These haplotype structures and their concomitant linkage disequilibrium among KIR genes suggest that more meaningful correlative data from studies of KIR genetics and complex disease may be achieved by measuring haplotypes of the KIR region in total. RESULTS: Towards that end, we developed a KIR haplotyping method that reports unambiguous combinations of KIR gene-content haplotypes, including both phase and copy number for each KIR. A total of 37 different gene content haplotypes were detected from 4,512 individuals and new sequence data was derived from haplotypes where the detailed structure was not previously available. CONCLUSIONS: These new structures suggest a number of specific recombinant events during the course of KIR evolution, and add to an expanding diversity of potential new KIR haplotypes derived from gene duplication, deletion, and hybridization.

Identification and characterization of 122 novel HLA alleles in bone marrow donors recruited to the Ezer Mizion Bone Marrow Donor Registry.

Between January 2018 and June 2021, the Ezer Mizion recruited 223,960 donors. All donors were typed for their HLA class I and II alleles at high resolution by Next Generation Sequencing techniques. Comparison between the sequences obtained from these donors and those in the IPD-IMGT/HLA Database, revealed 122 Novel HLA alleles that were found in 133 donors. Most of the alleles, 94 (77%) were identified in the HLA class I genes (30, 35, and 29 in HLA-A, -B, and -C, respectively), and 28 (23%) were detected in the HLA class II genes (9 in HLA-DRB1 and -DQB1 and 10 in -DPB1). Most of these novel alleles, 106 (86.9%) comprised single nucleotide variation (SNV), 9 (7.4%) present multiple amino acids variation, 4 and 3 were generated because of deletions and insertions, respectively. Ten of these novel alleles were seen to be null alleles.

Discovery of a novel HLA-C*04 null allele, HLA-C*04:279N, in a Singaporean individual.

A nucleotide mutation in codon 75 of HLA-C*04:01:01:01 results in a novel allele HLA-C*04:279N.

The MHC class I MICA gene is a histocompatibility antigen in kidney transplantation.

The identity of histocompatibility loci, besides human leukocyte antigen (HLA), remains elusive. The major histocompatibility complex (MHC) class I MICA gene is a candidate histocompatibility locus. Here, we investigate its role in a French multicenter cohort of 1,356 kidney transplants. MICA mismatches were associated with decreased graft survival (hazard ratio (HR), 2.12; 95% confidence interval (CI): 1.45-3.11; P < 0.001). Both before and after transplantation anti-MICA donor-specific antibodies (DSA) were strongly associated with increased antibody-mediated rejection (ABMR) (HR, 3.79; 95% CI: 1.94-7.39; P < 0.001; HR, 9.92; 95% CI: 7.43-13.20; P < 0.001, respectively). This effect was synergetic with that of anti-HLA DSA before and after transplantation (HR, 25.68; 95% CI: 3.31-199.41; P = 0.002; HR, 82.67; 95% CI: 33.67-202.97; P < 0.001, respectively). De novo-developed anti-MICA DSA were the most harmful because they were also associated with reduced graft survival (HR, 1.29; 95% CI: 1.05-1.58; P = 0.014). Finally, the damaging effect of anti-MICA DSA on graft survival was confirmed in an independent cohort of 168 patients with ABMR (HR, 1.71; 95% CI: 1.02-2.86; P = 0.041). In conclusion, assessment of MICA matching and immunization for the identification of patients at high risk for transplant rejection and loss is warranted.

Discovery of the novel HLA-C*06:195 allele in a Singaporean unrelated hematopoietic stem cell donor.

One nucleotide substitution in Codon 58 of HLA-C*06:02:01:01 results in a novel allele, HLA-C*06:195.

Identification of 33 novel HLA alleles in Arab potential bone marrow donors.

Between 2008 and April 2018, we recruited more than 37 000 potential Arab donors to the Hadassah Bone Marrow Donor Registry, all of whom were typed for high resolution HLA-A, -B, and -DRB1. In addition, more than 22% of them were also typed for their HLA-C and -DQB1 alleles. A comparison of the sequences obtained from these donors with the IPD-IMGT/HLA Database showed 33 novel alleles from five loci (HLA-A, -B, -C, -DR, -DQ). All of these novel HLA alleles were detected in the local Arab communities; 79% of these alleles have not been described in other groups yet and remain unique to the local Arab communities.

Discovery of HLA-DPB1*454:01 in a Singaporean Malay individual.

One nucleotide substitution in codon 85 of HLA-DPB1*04:01:01:01 results in a novel allele, HLA-DPB1*454:01.

HLA-DPB1*526:01 detected in two Singaporean Indian individuals.

One nucleotide substitution in codon 84 of HLA-DPB1*02:01:02:01 results in a novel allele, HLA-DPB1*526:01.

Novel HLA-DPB1*10:01:05 allele, identified by next-generation sequencing in a Saudi individual.

HLA-DPB1*10:01:05 differs from HLA-DPB1*10:01:01:01 by a single synonymous nucleotide substitution in exon 2, 38 G>A.

HLA-B*15:349:02, a novel variant of HLA-B*15, discovered in a Singaporean Malay bone marrow donor.

One nucleotide substitution in codon 112 of HLA-B*15:349:01 results in a novel allele, HLA-B*15:349:02.

The novel HLA-A*68:227 allele, identified by Next-Generation Sequencing in a Saudi individual.

HLA-A*68:227 differs from HLA-A*68:84 by two single nucleotide substitutions in codon 10 and 90.

HLA-DQB1*05:116, an HLA-DQB1*05 variant, detected in a Singaporean Chinese individual.

One nucleotide substitution in codon 87 of HLA-DQB1*05:02:01:01 results in a novel allele, HLA-DQB1*05:116.

Discovery of HLA-B*35:368, a novel HLA-B*35 variant, in a Singaporean Malay hematopoietic stem cell donor.

DNA substitutions from codons 69 to 71 of HLA-B*35:05:01:01 result in a novel allele, HLA-B*35:368.