The novel HLA-B*44 allele, HLA-B*44:220, identified by Single Molecule Real-Time DNA sequencing in a British Caucasoid male.

The genomic sequence of the novel HLA-B*44:220 allele identified in a British Caucasoid male.

Genomic sequence of the rare HLA-A*02:95 allele identified by sequence-based typing and cloning.

Genomic sequence of HLA-A*02:95 identified in an Anthony Nolan volunteer donor.

The novel KIR2DL1 allele, KIR2DL1*037, defined in the cell line SPO010 (IHW9036).

The novel KIR2DL1*037 allele discovered and characterised by single molecule real-time (SMRT) DNA sequencing.

KIR2DL1 allele sequence extensions and discovery of 2DL1*0010102 and 2DL1*0010103 alleles by DNA sequencing.

Full-length KIR2DL1 allele sequence extensions characterised by single molecule real-time (SMRT) DNA sequencing.

Single molecule real-time DNA sequencing of HLA genes at ultra-high resolution from 126 International HLA and Immunogenetics Workshop cell lines.

The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B-lymphoblastoid cell lines (B-LCLs) was established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B-LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing to HLA type 126 B-LCL, including the 107 International HLA and Immunogenetics Workshop (IHIW) cells, to ultra-high resolution. Amplicon sequencing of full-length HLA class I genes (HLA-A, -B and -C) and partial length HLA class II genes (HLA-DRB1, -DQB1 and -DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD-IMGT/HLA Database (Release 3.27.0, January 20, 2017), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n = 10) and changes in zygosity (n = 13), as well as previously unreported types (n = 34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four-field resolution typing of HLA-B and HLA-C. By improving and standardising the HLA typing of these B-LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing.

Recipient/donor HLA and CMV matching in recipients of T-cell-depleted unrelated donor haematopoietic cell transplants.

Improving haematopoietic cell transplantation outcomes by selection of an HLA-matched unrelated donor is best practice; however, donor selection by secondary characteristics is controversial. We studied 1271 recipients with haematological malignancies who underwent T-cell-depleted allografts and had complete data on HLA-matching status for six loci (HLA-A, -B, -C, -DRB1, -DQB1, -DPB1) and clinical outcome data. Five-year overall survival was 40.6%. HLA mismatching (at HLA-A, -B, -C, -DRB1, -DQB1) relative risk (RR) 1.22, 95% confidence interval (CI) 1.2-1.5, P=0.033 for 1 mismatch and RR 1.46, 95% CI 1.1-1.9, P=0.009 for >1 mismatch) and CMV mismatching (RR 1.37, 95% CI 1.2-1.6, P<0.001) were significantly associated with inferior survival. Donors aged <30 years showed a trend towards better survival. The multivariate model for mortality, combining CMV and HLA-match status, found an RR of 1.36 (95% CI 1.1-1.7, P=0.003) for HLA matched/CMV mismatched, an RR of 1.22 (95% CI 0.99-1.5, P=0.062) for HLA mismatched/CMV matched and an RR of 1.81 (95% CI 1.4-2.3, P=<0.001) for HLA/ CMV mismatched, compared with the HLA/CMV-matched recipients. These data suggest that HLA and CMV matching status should be considered when selecting unrelated donors and that CMV matching may abrogate the effect of an HLA mismatch.

Caspase-8 polymorphisms result in reduced Alemtuzumab-induced T-cell apoptosis and worse survival after transplantation.

Allo-SCT using unrelated donors is a curative treatment for patients with hematological disorders. The best donor is one matched for 10/10 HLA alleles, however studies have shown an additional survival benefit when considering other genetic factors. It has been shown that a six-nucleotide insertion/deletion polymorphism in the CASP8 gene promoter results in reduced susceptibility of T lymphocytes to undergo apoptosis. In 186 SCT recipients, we found a significantly better OS in those who received a transplant from a WT/WT donor compared with donors with a deletion (3 years: 52 vs 34%; P=0.03; multivariate analysis; RR 0.61; 95% CI 0.38-0.98, P=0.04). This was more marked when both the patient and the donor had a deletion (3 years OS: 62% compared with 36%, P=0.01). As the majority of these patients received Alemtuzumab during conditioning, we went on to analyze the in vitro effect of the polymorphism on Alemtuzumab-induced apoptosis. We showed statistically significantly higher percentages of apoptotic naïve CD4 (P<0.0005) and CD8 (P<0.0005) T cells in WT/WT donors in comparison with donors with a deletion. These data imply an unrecognized role for the CASP8 promoter polymorphism on survival following unrelated SCT particularly in the context of T-cell depletion with Alemtuzumab.

The degree of matching at HLA-DPB1 predicts for acute graft-versus-host disease and disease relapse following haematopoietic stem cell transplantation.

The importance of matching for HLA-DPB1 in unrelated donor haematopoietic stem cell (HSC) transplantation is little understood. Most transplant centres do not, currently, prospectively match for DPB1, but emerging data show that DPB1 matching does play a role in determining outcome. We studied the impact of HLA-DPB1 matching on outcome in 143 recipients of T-cell depletion transplants, who matched with their respective unrelated donors (allelic level) at HLA-A, -B, -C, -DRB1 and -DQB1. Of those matched at DPB1, 47.2% (17/36) developed acute graft-versus-host disease (aGvHD) as compared to 66.3% (55/83) of those who were mismatched. This led to a 19.1% (95% CI 0.1-38.3%) increase in the chance of developing aGvHD in mismatched patients (P=0.049). Relapse of the original disease occurred in 51 recipients; 23 of 37 (62%) matched at both DPB1 alleles, 28 of 82 (34%) were mismatched at one or two DPB1 alleles. Thus, there was a significantly higher relapse rate (P=0.0011) in transplant recipients who matched at both DPB1 alleles. In conclusion, a donor/recipient DPB1 match was associated with a significantly lower incidence of aGvHD and a significantly higher incidence of disease relapse. This study provides further evidence for an immunogenic role of HLA-DPB1 in HSC transplants.

Identification of amyloid-beta binding sites using an antisense peptide approach.

The amyloid-beta (A beta) peptide is a cytotoxic peptide implicated in the pathology of Alzheimer's disease (AD). Catalase and the endoplasmic reticulum A beta binding dehydrogenase (ERAB) are both inhibited by characterized fragments of the A beta peptide. In order to target such proteins it is essential to determine which components of these enzymes interact with A beta. This study reports the use of antisense peptide methodology to identify specific A beta-binding domains. Synthetic peptides corresponding to the regions of catalase and ERAB identified showed specific binding to A beta and also prevented A beta cytotoxicity. Antisense peptide methodology has identified A beta recognition sequences and may also be applied to the identification of novel A beta protein interactions to identify targets for use in the treatment of AD.

Diverging effects of HLA-DPB1 matching status on outcome following unrelated donor transplantation depending on disease stage and the degree of matching for other HLA alleles.

Disease stage and recipient/donor human leukocyte antigen (HLA) matching are important determinants of outcome in transplantation using volunteer-unrelated donors (VUD). Matching for HLA-A, -B, -C, -DRB1, -DQB1 is beneficial, whereas the importance of DPB1 matching is more controversial. The impact of HLA matching status may differ dependent on disease stage. We investigated the outcome according to the degree of HLA matching at 6 loci, in 488 recipients of predominantly T-cell depleted bone marrow VUD transplants for leukaemia. Survival was significantly better in 12/12-matched transplants in those with early leukaemia (5 years: 63 versus 41% in 10/10 matched, P=0.006), but not late stage disease. Conversely, within the HLA-mismatched group (< or =9/10), there was a significant survival advantage to DPB1 mismatching (5 years: 39 versus 21% in DPB1 matched, P=0.008), particularly in late leukaemia (P=0.01), persisting in multivariate analysis (odds ratio 0.478; 95% confidence interval 0.30, 0.75; P=0.001). These novel findings suggest that the best outcome for patients with early leukaemia, with a 10/10-matched donor, is achieved by matching for DPB1. Conversely, our results suggest that in patients receiving an HLA-mismatched graft, the outcome is significantly better if they are also mismatched for DPB1. We recommend validation of these results in independent datasets.

Sequence of a novel HLA-A*0301 intronic variant (A*03010103).

We report here the full-length sequence of a novel HLA-A*0301 allele, A*03010103, which differs from A*03010101 by a single nucleotide substitution (G>T) at position 492 within intron 2. The variant was originally identified by Reference Strand-mediated Conformational Analysis (RSCA) and was confirmed by cloning and sequencing. The difference in RSCA mobility between A*03010101 and A*03010103 demonstrates the sensitivity of RSCA to detect single nucleotide polymorphisms.

Polymorphisms in the TNFA gene promoter region show evidence of strong linkage disequilibrium with HLA and are associated with delayed neutrophil engraftment in unrelated donor hematopoietic stem cell transplantation.

Sustained myeloid engraftment is an important determinant of outcome in hematopoietic stem cell transplantation (HSCT). Human tumor necrosis factor (TNF)-alpha is encoded by a gene, TNFA, located in the class III region of the major histocompatibility complex on chromosome 6, flanked by the human leukocyte antigen (HLA) class I and II regions. A number of polymorphisms in the promoter region of the TNFA gene have been associated with increased production of TNF-alphain vivo. Additionally, raised TNF-alpha levels have been reported to have a detrimental effect on the outcome in HSCT, in particular on early complications such as acute graft vs host disease, failure to engraft, and transplant-related mortality. There is evidence of linkage disequilibrium (LD) between TNFA promoter polymorphisms and extended HLA haplotypes. We have genotyped 73 cell lines and 189 donor/recipient pairs (undergoing HSCT) for their TNFA polymorphism, all of which had been well characterized with respect to their HLA genes. We found evidence of strong LD between HLA genes and TNFA; however, there was also evidence for recombination events having taken place, as we found that a number of transplant pairs who were matched for their HLA haplotypes were not matched for their TNFA alleles. We analyzed early outcomes in the transplant recipients and found a significant delay in engraftment in those pairs where both donor and recipients possessed an AG allele (associated with higher TNF-alpha levels). Our results suggest a functional effect of TNFA polymorphisms on myeloid engraftment in unrelated HSCT.