Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers.

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3' region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3'-untranslated region (UTR). Disruption of the Pd-l1 3'-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3'-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow transplantation.

UNC-93 homolog B1 (UNC93B1) is a key regulator of toll-like receptors (TLRs), pattern recognition receptors that sense invading pathogens and manage the innate immune response and deliver them from the endoplasmic reticulum to their respective endosomal signaling compartments. Several types of TLRs are known to contribute to the inflammatory process after allogeneic hematopoietic stem cell transplantation (SCT), so UNC93B1 might play integral roles there. We investigated the influence of the UNC93B1 single-nucleotide polymorphism (SNP) rs308328 (T>C) on transplant outcomes in a cohort of 237 patients undergoing unrelated HLA-matched bone marrow transplantation (BMT) for hematologic malignancies through the Japan Marrow Donor Program. The donor UNC93B1 C/C genotype was associated with a better 3-year overall survival than the donor UNC93B1 C/T or T/T genotype. An analysis of the UNC93B1 rs308328 genotype may therefore be useful for selecting the donor, estimating the prognosis, and creating therapeutic strategies after allogeneic SCT.

Evolutionary basis of HLA-DPB1 alleles affects acute GVHD in unrelated donor stem cell transplantation.

HLA-DPB1 T-cell epitope (TCE) mismatching algorithm and rs9277534 SNP at the 3' untranslated region (3'UTR) in the HLA-DPB1 gene are key factors for transplant-related events in unrelated hematopoietic cell transplantation (UR-HCT). However, the association of these 2 mechanisms has not been elucidated. We analyzed 19 frequent HLA-DPB1 alleles derived from Japanese healthy subjects by next-generation sequencing of the entire HLA-DPB1 gene region and multi-SNP data of the HLA region in 1589 UR-HCT pairs. The risk of acute graft-versus-host disease (aGVHD) was analyzed in 1286 patients with single HLA-DPB1 mismatch UR-HCT. The phylogenetic tree constructed using the entire gene region demonstrated that HLA-DPB1 alleles were divided into 2 groups, HLA-DP2 and HLA-DP5. Although a phylogenetic relationship in the genomic region from exon 3 to 3'UTR (Ex3-3'UTR) obviously supported the division of HLA-DP2 and HLA-DP5 groups, which in exon 2 showed intermingling of HLA-DPB1 alleles in a non-HLA-DP2 and non-HLA-DP5-group manner. Multi-SNP data also showed 2 discriminative HLA-DPB1 groups according to Ex3-3'UTR. Risk of grade 2-4 aGVHD was significantly higher in patient HLA-DP5 group mismatch than patient HLA-DP2 group mismatch (hazard ratio, 1.28; P = .005), regardless of donor mismatch HLA-DP group. Regarding TCE mismatch, increasing risk of aGVHD in patient HLA-DP5 group mismatch and TCE-nonpermissive mismatch were observed only in patients with TCE-permissive mismatch and patient HLA-DP2 group mismatch, respectively. Evolutionary analysis revealed that rs9277534 represented a highly conserved HLA-DPB1 Ex3-3'UTR region and may provoke aGVHD differently to TCE mismatching algorithm, reflecting exon 2 polymorphisms. These findings enrich our understanding of the mechanism of aGVHD in HLA-DPB1 mismatch UR-HCT.

Identification of an HLA class I allele closely involved in the autoantigen presentation in acquired aplastic anemia.

To identify HLA alleles closely involved in the autoantigen presentation in acquired aplastic anemia (AA), we studied the HLA allelic loss frequencies of 312 AA patients, including 43 patients with loss of heterozygosity of 6p chromosome (6pLOH). An analysis of the HLA alleles contained in the lost haplotype revealed HLA-B*40:02 to be the most frequently lost allele. When we examined 28 AA (12 6pLOH[+] and 16 6pLOH[-]) patients with HLA-B*40:02 for the presence of leukocytes lacking HLA-B4002 (B4002-) using a new monoclonal antibody specific to this allele, B4002- granulocytes were detected not only in all 6pLOH(+) patients but also in 9 (56%) of the 16 6pLOH(-) patients. Furthermore, 10 (83%) of the 12 6pLOH(+) patients possessed 1.0% to 78% B4002- granulocytes that retained the HLA-A allele on the same haplotype (B4002-A+), suggesting the frequent coexistence of granulocytes that underwent mutations restricted to HLA-B*40:02 with 6pLOH(+) (B4002-A-) granulocytes. Deep sequencing of the HLA-B*40:02 of sorted B4002-A+ granulocytes revealed various somatic mutations, such as frameshift, nonsense, and splice site mutations, in all 15 patients studied. Surprisingly, missense mutations in the α-3 domain of HLA-B*40:02 that are not involved in the antigen presentation were detected exclusively in the B4002+ granulocytes of 3 patients possessing B4002- granulocytes. The markedly high prevalence of leukocytes lacking HLA-B4002 as a result of either 6pLOH or structural gene mutations, or both, suggests that antigen presentation by hematopoietic stem/progenitor cells to cytotoxic T cells via the HLA-B allele plays a critical role in the pathogenesis of AA.

Recipient ADAMTS13 Single-Nucleotide Polymorphism Predicts Relapse after Unrelated Bone Marrow Transplantation for Hematologic Malignancy.

Relapse remains a major obstacle to the survival of patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation. A disintegrin-like and metalloprotease with a thrombospondin type 1 motif (ADMATS13), which cleaves von Willebrand factor multimers into less active fragments, is encoded by the ADAMTS13 gene and has a functional single-nucleotide polymorphism (SNP) rs2285489 (C > T). We retrospectively examined whether ADAMTS13 rs2285489 affected the transplant outcomes in a cohort of 281 patients who underwent unrelated human leukocyte antigen (HLA)-matched bone marrow transplantation for hematologic malignancies. The recipient ADAMTS13 C/C genotype, which putatively has low inducibility, was associated with an increased relapse rate (hazard ratio [HR], 3.12; 95% confidence interval [CI], 1.25⁻7.77; P = 0.015), resulting in a lower disease-free survival rate in the patients with a recipient C/C genotype (HR, 1.64; 95% CI, 1.01⁻2.67; P = 0.045). Therefore, ADAMTS13 rs2285489 genotyping in transplant recipients may be a useful tool for evaluating pretransplantation risks.

New susceptibility variants to narcolepsy identified in HLA class II region.

Narcolepsy, a sleep disorder characterized by excessive daytime sleepiness, cataplexy and rapid eye movement sleep abnormalities, is tightly associated with human leukocyte antigen HLA-DQB1*06:02. DQB1*06:02 is common in the general population (10-30%); therefore, additional genetic factors are needed for the development of narcolepsy. In the present study, HLA-DQB1 in 664 Japanese narcoleptic subjects and 3131 Japanese control subjects was examined to determine whether HLA-DQB1 alleles located in trans of DQB1*06:02 are associated with narcolepsy. The strongest association was with DQB1*06:01 (P = 1.4 × 10(-10), odds ratio, OR = 0.39), as reported in previous studies. Additional predisposing effects of DQB1*03:02 were also found (P = 2.5 × 10(-9), OR = 1.97). A comparison between DQB1*06:02 heterozygous cases and controls revealed dominant protective effects of DQB1*06:01 and DQB1*05:01. In addition, a single-nucleotide polymorphism-based conditional analysis controlling for the effect of HLA-DQB1 was performed to determine whether there were other independent HLA associations outside of HLA-DQB1. This analysis revealed associations at HLA-DPB1 in the HLA class II region (rs3117242, P = 4.1 × 10(-5), OR = 2.45; DPB1*05:01, P = 8.1 × 10(-3), OR = 1.39). These results indicate that complex HLA class II associations contribute to the genetic predisposition to narcolepsy.

Genome-wide surveillance of mismatched alleles for graft-versus-host disease in stem cell transplantation.

Acute graft-versus-host disease (aGVHD) represents one of the major complications in allogeneic stem cell transplantation and is primarily caused by genetic disparity between the donor and recipient. In HLA-matched transplants, the disparity is thought to be determined by loci encoding minor histocompatibility antigens (minor H antigens), which are presented by specific HLA molecules. We performed a genome-wide association study (GWAS) to identify minor H antigen loci associated with aGVHD. A total of 500 568 single nucleotide polymorphisms (SNPs) were genotyped for donors and recipients from 1589 unrelated bone marrow transplants matched for HLA-A, -B, -C, -DRB1, and -DQB1, followed by the imputation of unobserved SNPs. We interrogated SNPs whose disparity between the donor and recipient was significantly associated with aGVHD development. Without assuming HLA unrestriction, we successfully captured a known association between HLA-DPB1 disparity (P = 4.50 × 10(-9)) and grade II-IV aGVHD development, providing proof of concept for the GWAS design aimed at discovering genetic disparity associated with aGVHD. In HLA-restricted analyses, whereby association tests were confined to major subgroups sharing common HLA alleles to identify putative minor H antigen loci, we identified 3 novel loci significantly associated with grade III-IV aGVHD. Among these, rs17473423 (P = 1.20 × 10(-11)) at 12p12.1 within the KRAS locus showed the most significant association in the subgroup, sharing HLA-DQB1*06:01. Our result suggested that a GWAS can be successfully applied to identify allele mismatch associated with aGVHD development, contributing to the understanding of the genetic basis of aGVHD.

Biological significance of HLA locus matching in unrelated donor bone marrow transplantation.

We hypothesized that the compatibility of each HLA loci between donor and patient induced divergent transplant-related immunologic responses, which attributed to the individualized manifestation of clinical outcomes. Here, we analyzed 7898 Japanese pairs transplanted with T-cell-replete marrow from an unrelated donor with complete HLA allele typing data. Multivariable competing risk regression analyses were conducted to evaluate the relative risk (RR) of clinical outcomes after transplantation. A significant RR of HLA allele mismatch compared with match was seen with HLA-A, -B, -C, and -DPB1 for grade III-IV acute graft-versus-host disease (GVHD), and HLA-C for chronic GVHD. Of note, only HLA-C and HLA-DPB1 mismatch reduced leukemia relapse, and this graft-versus-leukemia effect of HLA-DPB1 was independent of chronic GVHD. HLA-DRB1 and HLA-DQB1 double (DRB1_DQB1) mismatch was revealed to be a significant RR for acute GVHD and mortality, whereas single mismatch was not. Thus, the number of HLA-A, -B, -C, -DPB1, and DRB1_DQB1 mismatches showed a clear-cut risk difference for acute GVHD, whereas the number of mismatches for HLA-A, -B, -C, and DRB1_DQB1 showed the same for mortality. In conclusion, we determined the biological response to HLA locus mismatch in transplant-related immunologic events, and provide a rationale for use of a personalized algorithm for unrelated donor selection.

High-risk HLA alleles for severe acute graft-versus-host disease and mortality in unrelated donor bone marrow transplantation.

HLA molecules play an important role for immunoreactivity in allogeneic hematopoietic stem cell transplantation. To elucidate the effect of specific HLA alleles on acute graft-versus-host disease, we conducted a retrospective analysis using 6967 Japanese patients transplanted with T-cell-replete marrow from an unrelated donor. Using unbiased searches of patient and donor HLA alleles, patient and/or donor HLA-B*51:01 (patient: HR, 1.37,P<0.001; donor: HR, 1.35,P<0.001) and patient HLA-C*14:02 (HR, 1.35,P<0.001) were significantly associated with an increased risk of severe acute graft-versus-host disease. The finding that donor HLA-C*14:02 was not associated with severe acute graft-versus-host disease prompted us to elucidate the relation of these high-risk HLA alleles with patient and donor HLA-C allele mismatches. In comparison to HLA-C allele match, patient mismatched HLA-C*14:02 showed the highest risk of severe acute graft-versus-host disease (HR, 3.61,P<0.001) and transplant-related mortality (HR, 2.53,P<0.001) among all patient mismatched HLA-C alleles. Although patient HLA-C*14:02 and donor HLA-C*15:02 mismatch was usually KIR2DL-ligand mismatch in the graft-versus-host direction, the risk of patient mismatched HLA-C*14:02 for severe acute graft-versus-host disease was obvious regardless of KIR2DL-ligand matching. The effect of patient and/or donor HLA-B*51:01 on acute graft-versus-host disease was attributed not only to strong linkage disequilibrium of HLA-C*14:02 and -B*51:01, but also to the effect of HLA-B*51:01 itself. With regard to clinical implications, patient mismatched HLA-C*14:02 proved to be a potent risk factor for severe acute graft-versus-host disease and mortality, and should be considered a non-permissive HLA-C mismatch in donor selection for unrelated donor hematopoietic stem cell transplantation.

Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing.

BACKGROUND: HLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1. RESULTS: We developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template. CONCLUSIONS: In this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.

Immune-related pathways including HLA-DRB1(∗)13:02 are associated with panic disorder.

Panic disorder (PD) is an anxiety disorder characterized by panic attacks and anticipatory anxiety. Both genetic and environmental factors are thought to trigger PD onset. Previously, we performed a genome-wide association study (GWAS) for PD and focused on candidate SNPs with the lowest P values. However, there seemed to be a number of polymorphisms which did not reach genome-wide significance threshold due to their low allele frequencies and odds ratios, even though they were truly involved in pathogenesis. Therefore we performed pathway analyses in order to overcome the limitations of conventional single-marker analysis and identify associated SNPs with modest effects. Each pathway analysis indicated that pathways related to immunity showed the strongest association with PD (DAVID, P=2.08×10(-6); i-GSEA4GWAS, P<10(-3); ICSNPathway, P<10(-3)). Based on the results of pathway analyses and the previously performed GWAS for PD, we focused on and investigated HLA-B and HLA-DRB1 as candidate susceptibility genes for PD. We typed HLA-B and HLA-DRB1 in 744 subjects with PD and 1418 control subjects. Patients with PD were significantly more likely to carry HLA-DRB1(∗)13:02 (P=2.50×10(-4), odds ratio=1.54). Our study provided initial evidence that HLA-DRB1(∗)13:02 and genes involved in immune-related pathways are associated with PD. Future studies are necessary to confirm these results and clarify the underlying mechanisms causing PD.

A polymorphism in CCR1/CCR3 is associated with narcolepsy.

Etiology of narcolepsy-cataplexy involves multiple genetic and environmental factors. While the human leukocyte antigen (HLA)-DRB1*15:01-DQB1*06:02 haplotype is strongly associated with narcolepsy, it is not sufficient for disease development. To identify additional, non-HLA susceptibility genes, we conducted a genome-wide association study (GWAS) using Japanese samples. An initial sample set comprising 409 cases and 1562 controls was used for the GWAS of 525,196 single nucleotide polymorphisms (SNPs) located outside the HLA region. An independent sample set comprising 240 cases and 869 controls was then genotyped at 37 SNPs identified in the GWAS. We found that narcolepsy was associated with a SNP in the promoter region of chemokine (C-C motif) receptor 1 (CCR1) (rs3181077, P=1.6×10(-5), odds ratio [OR]=1.86). This rs3181077 association was replicated with the independent sample set (P=0.032, OR=1.36). We measured mRNA levels of candidate genes in peripheral blood samples of 38 cases and 37 controls. CCR1 and CCR3 mRNA levels were significantly lower in patients than in healthy controls, and CCR1 mRNA levels were associated with rs3181077 genotypes. In vitro chemotaxis assays were also performed to measure monocyte migration. We observed that monocytes from carriers of the rs3181077 risk allele had lower migration indices with a CCR1 ligand. CCR1 and CCR3 are newly discovered susceptibility genes for narcolepsy. These results highlight the potential role of CCR genes in narcolepsy and support the hypothesis that patients with narcolepsy have impaired immune function.

Significant association of KIR2DL3-HLA-C1 combination with cerebral malaria and implications for co-evolution of KIR and HLA.

Cerebral malaria is a major, life-threatening complication of Plasmodium falciparum malaria, and has very high mortality rate. In murine malaria models, natural killer (NK) cell responses have been shown to play a crucial role in the pathogenesis of cerebral malaria. To investigate the role of NK cells in the developmental process of human cerebral malaria, we conducted a case-control study examining genotypes for killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen (HLA) class I ligands in 477 malaria patients. We found that the combination of KIR2DL3 and its cognate HLA-C1 ligand was significantly associated with the development of cerebral malaria when compared with non-cerebral malaria (odds ratio 3.14, 95% confidence interval 1.52-6.48, P = 0.00079, corrected P = 0.02). In contrast, no other KIR-HLA pairs showed a significant association with cerebral malaria, suggesting that the NK cell repertoire shaped by the KIR2DL3-HLA-C1 interaction shows certain functional responses that facilitate development of cerebral malaria. Furthermore, the frequency of the KIR2DL3-HLA-C1 combination was found to be significantly lower in malaria high-endemic populations. These results suggest that natural selection has reduced the frequency of the KIR2DL3-HLA-C1 combination in malaria high-endemic populations because of the propensity of interaction between KIR2DL3 and C1 to favor development of cerebral malaria. Our findings provide one possible explanation for KIR-HLA co-evolution driven by a microbial pathogen, and its effect on the global distribution of malaria, KIR and HLA.

Single nucleotide polymorphisms and outcome risk in unrelated mismatched hematopoietic stem cell transplantation: an exploration study.

Genetic risk factors contribute to adverse outcome of hematopoietic stem cell transplantation (HSCT). Mismatching of the HLA complex most strongly determines outcomes, whereas non-HLA genetic polymorphisms are also having an impact. Although the majority of HSCTs are mismatched, only few studies have investigated the effects of non-HLA polymorphisms in the unrelated HSCT and HLA-mismatched setting. To understand these effects, we genotyped 41 previously studied single nucleotide polymorphisms (SNPs) in 2 independent, large cohorts of HSCT donor-recipient pairs (n = 460 and 462 pairs) from a homogeneous genetic background. The study population was chosen to pragmatically represent a large clinically homogeneous group (acute leukemia), allowing all degrees of HLA matching. The TNF-1031 donor-recipient genotype mismatch association with acute GVHD grade 4 was the only consistent association identified. Analysis of a subgroup of higher HLA matching showed consistent associations of the recipient IL2-330 GT genotype with risk of chronic GVHD, and the donor CTLA4-CT60 GG genotype with protection from acute GVHD. These associations are strong candidates for prediction of risk in a clinical setting. This study shows that non-HLA gene polymorphisms are of relevance for predicting HSCT outcome, even for HLA mismatched transplants.

The role of alloantibodies against human platelet antigen-15 in multiply platelet transfused patients.

BACKGROUND: Several studies have documented the role of antibodies against human platelet (PLT) antigen (HPA)-15 in alloimmune-mediated thrombocytopenia including neonatal alloimmune thrombocytopenia, PLT transfusion refractoriness (PTR), and posttransfusion purpura in Caucasian persons. However, the relevance of anti-HPA-15 in PTR among the Japanese population is still unclear. STUDY DESIGN AND METHODS: The sera of 305 multiply PLT transfused (MPT) patients, previously investigated for the presence of human leukocyte antigen (HLA) and HPA antibodies by mixed passive hemagglutination, were reexamined for the presence of HPA-15 alloantibodies, using the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) technique. RESULTS: Among the 305 MPT samples, antibodies against HPA-15 alloantigen was detected in seven (2.3%), two (0.66%) being anti-HPA-15a and five (1.64%) being anti-HPA-15b. Additionally, one case of CD109 panreactive antibody was found (0.33%). Among them, one aplastic anemia patient with blood group O developed multispecific anti-HLA and anti-HPA-15b alloantibody after MPTs. However, transfusion with HLA-matched PLTs of blood group AB did not result in adequate PLT count increment. Analysis of the possible influence of immune anti-A and anti-B by the MAIPA assay resulted negative, indicating that anti-HPA-15b is responsible for the refractory state in this patient. CONCLUSION: In this study, we found alloimmunization against HPA-15a and -15b in Japanese populations and demonstrated the relevance of these antibodies in a patient with PTR.

Significance of ethnicity in the risk of acute graft-versus-host disease and leukemia relapse after unrelated donor hematopoietic stem cell transplantation.

The significance of patient and donor ethnicity on risk of acute graft-versus-host disease (GVHD) and disease relapse after unrelated donor hematopoietic cell transplantation (HCT) is not known. A total of 4335 patient-donor pairs from the International Histocompatibility Working Group in HCT met the following 3 criteria: (1) HLA-A, -B, -C, -DRB1, and -DQB1 allele matched donor, (2) diagnosis of leukemia, and (3) non-T cell depleted GVHD prophylaxis. Posttransplantation risks of acute GVHD and leukemia relapse were defined in Asian/Pacific Islander, white, African American, Hispanic, and Native American patients that underwent transplantation from donors with the same self-described background. Asian patients had a significantly lower incidence of acute GVHD (Japanese patients: 40.0% grades II to IV and 15.3% grades III to IV; non-Japanese Asian patients: 42.1% grades II to IV and 15.7% grades III to IV) compared with white patients (56.5% grades II to IV and 22.6% grades III to IV) (P < .001). The hazard ratio of acute GVHD for white patients was significantly higher than for Japanese patients. Unexpectedly, the hazard ratio of leukemia relapse in white patients with early disease status was also significantly higher than that in Japanese patients. These results provide a platform for future investigation into the genetic factors for unrelated donor HCT and clinical implications of diverse ethnic background.

Exome sequencing identifies novel rheumatoid arthritis-susceptible variants in the BTNL2.

The butyrophilin-like protein 2 gene (BTNL2) within the class III region of the major histocompatibility complex genomic region was identified as a rheumatoid arthritis (RA) susceptibility gene by exome sequencing (19 RA cases) with stepwise filtering analysis, and then validated by Sanger sequencing and association analysis using 432 cases and 432 controls. Logistic regression of the Sanger-sequenced single-nucleotide variants in an association study of 432 cases and 432 controls showed that 12 non-synonymous single-nucleotide polymorphisms (SNPs) in BTNL2 were significantly associated with RA. The lowest P-values were obtained from three SNPs, rs41521946, rs28362677 and rs28362678, which were in absolute linkage disequilibrium: P=4.55E-09, odds ratio=1.88, 95% confidence interval=1.52-2.33. The BTNL2 locates on chromosome 6 between HLA-DRB1 and NOTCH4, and is 170 kb apart from these two genes. Although DRB1 and NOTCH4 were reported to be RA-susceptible, the three BTNL2 SNPs retained significant association with RA when evaluated by the logistic regression with the adjustment for RA-susceptible HLA-DRB1 alleles in Japanese or rs2071282-T in NOTCH4: P=0.0156 and P=0.00368, respectively. These results suggest that the three non-synonymous SNPs in BTNL2 confer RA risk independently from HLA-DRB1 and NOTCH4.

Frequent loss of HLA alleles associated with copy number-neutral 6pLOH in acquired aplastic anemia.

Idiopathic aplastic anemia (AA) is a common cause of acquired BM failure. Although autoimmunity to hematopoietic progenitors is thought to be responsible for its pathogenesis, little is known about the molecular basis of this autoimmunity. Here we show that a substantial proportion of AA patients harbor clonal hematopoiesis characterized by the presence of acquired copy number-neutral loss of heterozygosity (CNN-LOH) of the 6p arms (6pLOH). The 6pLOH commonly involved the HLA locus, leading to loss of one HLA haplotype. Loss of HLA-A expression from multiple lineages of leukocytes was confirmed by flow cytometry in all 6pLOH(+) cases. Surprisingly, the missing HLA-alleles in 6pLOH(+) clones were conspicuously biased to particular alleles, including HLA-A*02:01, A*02:06, A*31:01, and B*40:02. A large-scale epidemiologic study on the HLA alleles of patients with various hematologic diseases revealed that the 4 HLA alleles were over-represented in the germline of AA patients. These findings indicate that the 6pLOH(+) hematopoiesis found in AA represents "escapes" hematopoiesis from the autoimmunity, which is mediated by cytotoxic T cells that target the relevant auto-antigens presented on hematopoietic progenitors through these class I HLAs. Our results provide a novel insight into the genetic basis of the pathogenesis of AA.

[HLA genotyping for molecular epidemiological analysis of humans].

The major histocompatibility complex (MHC) gene family in humans is termed the human leukocyte antigen (HLA) locus, and is one of the most polymorphic loci in the human genome. HLA and related genes play many important roles in the immune system. HLA matching is critical for the success of organ and hematopoietic stem cell transplantation. Additionally, HLA genes and proteins are associated with autoimmunity, immunity to infections, tumor development, and adverse drug reactions (ADR). For matching donor and recipient HLA types and to understand the role played by the HLA in disease development, lymphocyte cytotoxicity tests (LCT) were developed by Dr. Terasaki in the 1960s. This serological typing method, which was in use until recently, is now no longer used and has been replaced by DNA-based typing methods such as PCR-SSP (sequence-specific primers), PCR-rSSO(reverse sequence-specific oligonucleotide probes), and PCR-SBT (sequence-based typing). In the future, the current HLA-typing methods will be replaced by faster, more detailed and more cost-effective methods such as next-generation sequencing (NGS). In this review, we focus on the past and present methods of HLA typing, and the role of the HLA in various diseases.

Impact of highly conserved HLA haplotype on acute graft-versus-host disease.

Although the effects of human leukocyte antigen (HLA) locus matching on clinical outcome in unrelated hematopoietic stem cell transplantations have been characterized, the biologic implications of HLA haplotypes have not been defined. We demonstrated the genetic fixity of Japanese conserved extended haplotypes by multi-single nucleotide polymorphism analysis in 1810 Japanese donor-recipient pairs matching with HLA-A, -B, -C, -DRB1, and -DQB1 alleles. Three major Japanese conserved extended haplotypes (named HP-P1, HP-P2, and HP-P3) were essentially completely conserved at least in the 3.3-Mb HLA region from HLA-A to -DPB1, and extended far beyond HLA-A. The risk of acute graft-versus-host disease (GVHD) of these HLA haplotypes was assessed with multivariate Cox regression in 712 patients transplanted from HLA fully (HLA-A, B, C, DRB1, DQB1, and DPB1) matched unrelated donors. HP-P2 itself reduced the risk of grade 2 to 4 acute GVHD (hazard ratio [HR] = 0.63; P = .032 compared with HP-P2-negative), whereas HP-P3 tended to increase the risk (HR = 1.38; P = .07). Among 381 patients with HP-P1, HP-P1/P3 (HR = 3.35; P = .024) significantly increased the risk of acute GVHD compared with homozygous HP-P1. This study is the first to demonstrate that a genetic difference derived from HLA haplotype itself is associated with acute GVHD in allogeneic hematopoietic stem cell transplantation.