DRB1 Loci Research Articles

Deriving HLA genotyping from whole genome sequencing data using Omixon HLA twin(tm) in G3’s global clinical study

Aim Identify the degree of concordance and the ambiguities when DNA samples are analyzed using two HLA genotyping systems; Whole Genome Sequencing (WGS) data analyzed by Omixon HLA Target software and genotyping by the Holotype HLA typing system. Methods WGS data were generated of 235 human samples from G3’s GLOBAL study (NCT01738828) using paired 100 bps long HiSeq Illumina ∼30× coverage and analyzed by the Omixon HLA Target software. Genotyping was also performed for the same samples using the Holotype HLA typing system, whereby the generated libraries were sequenced on the Illumina MiSeq using the 250 bps paired-end sequencing protocol. Data were analyzed by the Omixon HLA Twin software. Concordance at the three-field level between the two approaches was assessed for each of the HLA-A, B, C, DQB1 and DRB1 loci. Ambiguities were also determined for each of the typing methods. Results A pairwise comparison between the two NGS-based methods shows that in average they were 97.2% concordant at the three-fields level (HLA-A: 450/470 95.7%, HLA-B: 464/470 98.7%, HLA-C: 464/470 98.7%, HLA-DQB1: 438/470 93.2%, HLA-DRB1 467/468 99.8%). Most of the discrepancies were due to allele dropout in the WGS sample or systematic errors of the software. The 41 non-systematic mistypings were due to either insufficient coverage of the WGS data or algorithmic problems. The Holotype HLA system did not generate any ambiguities for the HLA-A, -B, -C and HLA-DQB1 loci at the three-fields typing level. However, as intron 1 and exon 1 of DRB1 are not targeted, 1.4% of ambiguities were generated for the DRB1 locus.The WGS typing considers only the exons of the reference database, leading to phase ambiguities and overall ambigous typings of 9.8%, 4.3%, 7.7%, 8.3% and 3.4% for HLA-A, B, C, DQB1 and DRB1 respectively. Conclusions HLA genotyping using WGS data derived through next-generation sequencing and analyzed by the Omixon HLA Twin software is possible at 97.2% accuracy level. Future improvements of the software addressing ambiguities and remaining systematic errors can make HLA typing from WGS data more reliable. S. Juhos: Employee; Company/Organization; Omixon Ltd. T. V a g o : Employee; Company/Organization; Omixon Ltd. D. Ferriola: Employee; Company/Organization; Children’s Hospital of Philadelphia. J. Duke: Employee; Company/Organization; Children’s Hospital of Philadelphia. S. V o r o s: Employee; Company/Organization; Global Genomics Group. B.O. Brown: Employee; Company/Organization; Global Genomics Group. I. Marvasty: Employee; Company/Organization; Global Genomics Group. T. Hague: Employee; Company/Organization; Omixon Ltd. D. Monos: Employee; Company/Organization; Children’s Hospital of Philadelphia.

Commom, well-documented and rare HLA alleles observed in the brazilian bone marrow registry

Aim We intend to characterize alleles with 5 or more counts in non related donors of the Brazilian Bone Marrow Registry (REDOME) that are not listed in common and well documented (CWD) alleles version 2. Methods Identification and comparison of alleles and NMDP alleles codes which first allele in the subtype are not in CWD version 2 from the REDOME data base at February 15, 2015. Results We identified 30,395 HLA typings with 1575 alleles or NMDP alelle codes of not common or not well documented among the 3,227,387 donors. A total of 1213 donor registries corresponding to 158 alleles in A locus, 306 in B locus, 47 in C locus, 136 in DRB1 locus and 37 in DQB1 locus had less of 5 counts. In a few cases (28/283) it was possible to have access to the outputs and from these, 11 were probably misinterpreted results, although in one case A∗02:90 was also reported in Chinese donor registry. We also identified 259 alleles with 5–25 counts, 116 alleles with 26–100 counts, 29 alleles with 101–500, and 4 with more than 500. Eighty of these alleles or NMDP allele were most likely misinterpretation due to SSO bead adjustments corresponding to 9582 donors. Actual SSO output reviewed interpretation and SBT confirmed typings are show in Table 1. Conclusion Sequencing based typings were on going to confirm these Brazilian CWD HLA alleles and are displayed at www.imunogenetica.org (in Portuguese). Download : Download full-size image

Uncommon DR7-DR53-DQ haplotypes in four canadian subjects

Aim To describe uncommon DR7-DR53-DQ haplotypes in four Canadian subjects that deviate from the frequent common association between DR7, DR53 and DQ antigens. Methods Medium resolution HLA typing for HLA- DRB1, DRB3/4/5 and DQB1 loci were performed using Luminex based SSO kits (One Lambda Labtype). A high resolution typing using SSP for DRB4 (Olerup SSP kit) and SBT for DRB1 and DQB1 (Life Technologies) were further performed to evaluate these uncommon haplotypes. Results We detected presence of DRB4 ∗ 01:01 instead of more frequent DRB4 ∗ 01:03N in association with the DR7 and DQ9 haplotype in two Canadian subjects. Furthermore, we also detected DRB4 null allele in association with DR7 and DQ2 in a kidney transplant recipient and a bone marrow patient (Table 1). Conclusion Here, we report two cases of DRB1∗07, DRB4∗01, DQB1∗03:03 haplotype and two cases of DRB4∗01:03N null allele in association with DRB1 ∗ 07:01 and DQB1 ∗ 02:02 haplotype . Our findings are in disparity to reported common linkages in which DR7- DQ9 haplotype is commonly seen in association with the null allele DRB4 ∗ 01:03:01:02N . There are 15 alleles of DRB4; 12 of them produce an expressed DR53 antigen and 3 resulting in null alleles. The presence of these rare haplotypes might be due to unidirectional or bidirectional homologous recombination event that happened between the DR7, DR53, DQ2 and the DR7, DR53- null, DQ9 haplotypes. This is supported by the presence of reported DR7-DQ2 haplotype with a DRB4 null allele. It might also be due to point mutation in DRB4∗01:03:01 changing the splice site in case of DR7, DR53null, DQ2 haplotype. Understanding the genetics and expression of DRB4 gene is of clinical relevance in the hematopoetic stem cell transplant, both GVHD and graft rejection, in solid organ transplant especially effect of donor specific antibodies, and in disease association with autoimmune disorders, such as celiac, Crohn’s disease, myasthenia gravis, rheumatoid arthritis, Hashimoto’s thyroiditis, vitiligo, primary biliary cirrhosis and ALL in children. Download full-size image

HLA typing by NGS in the clinical lab: our one and a half years experience

Aim We present our experience using the Holotype™ Kit by Omixon for next generation sequencing (NGS) of HLA genes in a clinical setting for one and a half years, starting December 2013. Methods Samples were prepared using the X4 Holotype™ kit by Omixon for up to 7 loci (full length HLA-A, B, C, DQA1 and DQB1, intron 1 to intron 4 of DRB1, and intron 1–3’ UTR of DPB1) and were sequenced in both an individual and pooled amplicon approach using 2 × 251 bp reads on the Illumina MiSeq. Samples were also characterized in parallel with a second method (Sanger SBT, SSO, or SSP) to confirm the genotyping results by NGS and identify possible causes of discrepancies, if any. Two analysis programs were used to genotype the NGS data, Target by Omixon (v 1.7/1.8) and NGSengine by GenDX (v 1.3/1.6). Results A total of 1046 samples have been sequenced: 497 (47%) related to bone marrow transplant cases, 250 (24%) related to solid organ transplantation, and 299 (29%) samples being either proficiency, outreach or disease association studies. Approximately 975 typings were performed for HLA-A, B, C, DRB1 and DQB1, and 600 typings for DQA1 and DPB1. On average, one sequencing run was performed per week with 12 samples on the MiSeq for a total of 83 runs utilizing both nano and full flow cells. At best, the turnaround time from amplification to analysis completion was 4 days, but averages 10 days due to the laboratory workflow and a single sequencing run per week. Concordance was 99.7% between NGS and secondary typing methods (SBT/SSP/SSOP). Discordance was due to incorrect or incomplete (ambiguous) SBT/SSP typing, allele imbalance, NGS genotyping software errors, incorrect reporting of results and new alleles. Two genotyping programs were used for analysis to safeguard against systematic analysis errors and agree for 94.8% of the best match allele calls. Ambiguous results are most common at the DPB1 locus due to a lack of phasing between exons 2 and 3 or the unsequenced exon 1 (17% of alleles) and the DRB1 locus due to not sequencing exon 1 (3.8% of alleles). No ambiguities were detected among the other loci. Conclusions We have genotyped over 1000 samples using NGS, with a very high concordance rate with other HLA typing methods and low ambiguity rates for DRB1 and DPB1 as described above. This technology is therefore very appropriate for HLA typing in a clinical setting. D. Monos: Grant/Research Support; Company/Organization; Omixon. 7. Other (Identify); Company/Organization; Royalty from Omixon.

Concordance of next generation sequence-based and sequence specific oligonucleotide probe-based HLA-DRB1 genotyping

Next generation sequencing (NGS) of clonally amplified DNA, using Roche 454 technology, was used to genotype HLA-DRB1, DRB3, DRB4, and DRB5 loci (exon 2 only) from a set of 993 samples from newborns with maternally-reported African American ancestry. DRB1 exon 2 was genotyped previously on the same sample set using sequence-specific oligonucleotide probe (SSOP) technology. Comparison of the genotype calls from both methods indicated concordance of 92.3%. Some discordance was expected due to the higher resolution of NGS data, compared to SSOP data. This resulted from selection of the incorrect allele from the ambiguity string produced by SSOP genotyping. Of 76 discordant genotypes, only three were due to resolution of ambiguity with the NGS method. The low percent of changes due to the increased resolution of the NGS method instills confidence in the overall value of previous data genotyped with moderate resolution methods, i.e., the vast majority of alleles present in a population are those that are detectable at moderate resolution. The remaining 73 discordant genotypes resulted from preventable errors in sample handling, data interpretation, and data entry. These results underscore the potential for error that can result from factors such as low quality genomic DNA, manual data entry, and interpretation of marginal genotyping results. Optimization of genomic DNA quality, automation of genotyping steps wherever possible, and use of the highest resolution technology available can lead to dramatic improvements in HLA genotype data quality. NGS-based methodology generated data of superior quality and accuracy compared to the SSOP system.

HLA alleles association and recurrent spontaneous abortion in a São Luis/Maranhão population, in Brazilian Northeastern region

To investigate the association of the HLA-A, -B and -DRB1 alleles with the occurrence of Recurrent Spontaneous Abortion. A case-control study of 200 women aged 18 to 35 years, consisting of a convenience sample of 100 women who had idiopathic recurrent spontaneous abortion and 100 women without abortion and with two or more children. Peripheral blood genomic DNA was extracted from 500l of Buffy Coat stored at -20°C. HLA typing was performed by the PCR-SSOP method (Polymerase Chain Reaction - Specific Sequence of Oligonucleotides Probes, One Lambda(r), CA, USA). The regions of the amplified DNA were exon 2 and 3 for the A and B loci and only exon 3 for the DRB1 locus. The HLA FUSIONTM program (One Lambda, Canoga Park, CA, USA, version 3.0) was used for HLA-A, HLA-B and HLA-DRB1 genotyping. Absolute frequencies and percentages and calculation of mean and standard deviation were used for standard statistical analysis. The qualitative variables were compared by the χ2 test with Yates correction or by Fisher's exact test. The odds ratio with the 95%CI was used for the comparisons, with the level of significance set at p<0.05. The frequency of the A*34 allele was significantly higher in the case group compared to control (4.0 versus 0.5%; p<0.05). Alleles A*24 (6.0 versus 12.5%; p<0.05) and B*35 (8.0 versus 20.5%; p<0.05) were significantly less frequent in the case group. Among the class II alleles, DRB1*03 showed a slightly higher frequency in the case group (11.0 versus 5.5%, p = 0.056). It was shown that the HLA-A*34 allele is a risk factor for recurrent spontaneous abortion, while the HLA-A*24 and HLA-B*35 alleles are associated with protection, and no allele of the DRB1 locus was associated with RSA.

Genetic association study of the HLA class II alleles DRB1, DQA1, and DQB1 in patients with pharmacoresistant temporal lobe epilepsy associated with mesial hippocampal sclerosis

PurposeTemporal lobe epilepsy (TLE) is the most common variety of focal epilepsy among adults. The neuroinflammatory mechanisms of epilepsies may be involved in the genesis of seizures and refractory epilepsies, particularly in the case of progressive syndromes such as TLE associated with mesial hippocampal sclerosis (TLE-HS). The goal of the present study is investigate the genetic profile of susceptibility of individuals with TLE-HS by analyzing the possible association of TLE-HS with human leukocyte antigen (HLA) DRB1, DQA1 and DQB1 alleles. MethodsPeripheral blood samples were collected from 42 individuals with pharmacoresistant TLE-HS and 89 healthy controls. The typing of the HLA class II alleles from DRB1, DQB1, and DQA1 loci were analyzed using sequence-specific primer-polymerase chain reaction (SSP-PCR) and identified through sequencing. Statistical analysis of relative allele frequencies was performed using an Excel spreadsheet; p-value, relative risk (RR), and odds ratio (OR) were calculated using the software Epi Info 6.0. p-values <0.05 following Bonferroni's method correction were considered statistically significant. ResultsHLA-DRB1*13:02 was the only allele with a statistically significant difference (p=0.01) in frequency between patients and controls. However, the significance was lost following Bonferroni's method correction (p=0.44). The remainder of the alleles in the HLA-DRB1, HLA-DQB1 and HLA-DQA1 regions did not exhibit any significant association. ConclusionThe allele HLA DRB1*13:02 has exhibited a tendency to behave as a susceptibility factor for TLE-HS.

Development and validation of a sample sparing strategy for HLA typing utilizing next generation sequencing

We report the development of a general methodology to genotype HLA class I and class II loci. A Whole Genome Amplification (WGA) step was used as a sample sparing methodology. HLA typing data could be obtained with as few as 300 cells, underlining the usefulness of the methodology for studies for which limited cells are available. The next generation sequencing platform was validated using a panel of cell lines from the International Histocompatibility Working Group (IHWG) for HLA-A, -B, and -C. Concordance with the known, previously determined HLA types was 99%. We next developed a panel of primers to allow HLA typing of alpha and beta chains of the HLA DQ and DP loci and the beta chain of the DRB1 locus. For the beta chain genes, we employed a novel strategy using primers in the intron regions surrounding exon 2, and the introns surrounding exons 3 through 4 (DRB1) or 5 (DQB1 and DPB1). Concordance with previously determined HLA Class II types was also 99%. To increase throughput and decrease cost, we developed strategies combining multiple loci from each donor. Multiplexing of 96 samples per run resulted in increases in throughput of approximately 8-fold. The pipeline developed for this analysis (HLATyphon) is available for download at https://github.com/LJI-Bioinformatics/HLATyphon.

Identification of Disease-Promoting HLA Class I and Protective Class II Modifiers in Japanese Patients with Familial Mediterranean Fever.

ObjectivesThe genotype-phenotype correlation of MEFV remains unclear for the familial Mediterranean fever (FMF) patients, especially without canonical MEFV mutations in exon 10. The risk of FMF appeared to be under the influence of other factors in this case. The contribution of HLA polymorphisms to the risk of FMF was examined as strong candidates of modifier genes.MethodsGenotypes of HLA-B and -DRB1 loci were determined for 258 mutually unrelated Japanese FMF patients, who satisfied modified Tel-Hashomer criteria, and 299 healthy controls. The effects of carrier status were evaluated for the risk of FMF by odds ratio (OR). The HLA effects were also assessed for clinical forms of FMF, subsets of FMF with certain MEFV genotypes and responsiveness to colchicine treatment.ResultsThe carriers of B*39:01 were increased in the patients (OR = 3.25, p = 0.0012), whereas those of DRB1*15:02 were decreased (OR = 0.45, p = 0.00050), satisfying Bonferroni’s correction for multiple statistical tests (n = 28, p<0.00179). The protective effect of DRB1*15:02 was completely disappeared in the co-existence of B*40:01. The HLA effects were generally augmented in the patients without a canonical MEFV variant allele M694I, in accordance with the notion that the lower penetrance of the mutations is owing to the larger contribution of modifier genes in the pathogenesis, with a few exceptions. Further, 42.9% of 14 colchicine-resistant patients and 13.5% of 156 colchicine-responders possessed B*35:01 allele, giving OR of 4.82 (p = 0.0041).ConclusionsThe differential effects of HLA class I and class II polymorphisms were identified for Japanese FMF even in those with high-penetrance MEFV mutations.

HLA associations in South Asian multiple sclerosis

Background: Previous efforts to identify Human Leukocyte Antigen (HLA) gene associations with multiple sclerosis (MS) in the South Asian population have been underpowered. Aim: To identify the primary HLA class II alleles associated with MS in Indians. Methods: We typed HLA-DRB1, -DQA1 and -DQB1 in 419 patients and 451 unrelated controls by polymerase chain reaction using sequence specific oligonucleotide probes (PCR-SSOP). Results: At the gene level DRB1 showed significant evidence of association (p=0.0000012), DQA1 showed only marginal evidence of association (p=0.04) and there was no evidence for association at DQB1 (p=0.26). At the DRB1 locus association is confirmed with the *15:01 (p=0.00002) and the *03 (p=0.00005) alleles. Conclusion: Our study confirms that the risk effects attributable to the HLA- DRB1*15:01and DRB1*03 alleles seen in Europeans are also seen in Indians. The absence of any evidence of association with DQB1 alleles reflects the lower linkage disequilibrium between DQB1 alleles and DRB1 risk alleles present in this population, and illustrates the potential value of fine mapping signals of association in different ethnic groups.

The CIITA genetic polymorphism rs4774*C in combination with the HLA-DRB1*15:01 allele as a putative susceptibility factor to multiple sclerosis in Brazilian females.

The objective of this study was to investigate the association between the HLA alleles at the DQA1, DQB1 and DRB1 loci, the CIITA genetic polymorphisms -168A/G and +1614G/C, and susceptibility to multiple sclerosis (MS) in a sample from Rio de Janeiro State, Brazil. Furthermore, we wished to determine whether any of these associations might be more significant in women compared with men. DNA samples from 52 relapsing-remitting MS (RRMS) patients and 126 healthy controls matched for sex and age were analyzed. We identified a significant HLA-DRB1*15:01-MS association that was female-specific (Odds Ratio (OR) = 4.78; p = 0.001). Furthermore, we observed that the +1614G/C mutation in combination with the HLA-DRB1*15:01 allele increased susceptibility to MS in females (OR = 4.55; p = 0.01). Together, these findings highlight the polygenic nature of MS.

HLA-A, -B, -C, and -DRB1 genotyping of 1000 Northern Irish individuals from Belfast, Northern Ireland in the United Kingdom

One thousand individuals from Belfast, Northern Ireland were genotyped at the HLA-A, -B, -C and -DRB1 loci using sequence-specific oligonucleotide probe methods. HLA-A locus genotypes display a minor Hardy–Weinberg (HW) deviation (p=0.0375); HLA-B, -C and -DRB1 genotypes are consistent with expected HW proportions. These genotype data are available in the Allele Frequencies Net Database under identifier AFND 1243.

Clinical Outcomes of HLA-DPB1 Mismatches in 10/10 HLA-Matched Donor-Recipient Pairs Undergoing Allogeneic Stem Cell Transplant

Matching for HLA-DPB1 lowers relapse risk in allogeneic hematopoietic stem cell transplantation (ASCT), but this locus is not in linkage disequilibrium with the remainder of the HLA genes, making matching more difficult. Recent studies have suggested that after classifying HLA-DPB1 mismatches based on T-cell epitope group, avoiding non-permissive mismatches leads to improved survival. We tested this hypothesis in our patient population. We conducted a retrospective analysis of 78 patients who underwent ASCT at the Mayo Clinic 1/1/2008 - 12/21/2010. Patients with unrelated volunteer donors were matched for HLA-A, B, C, DRB1 and DQB1 loci (10/10) at the allele level without consideration of the HLA-DPB1 locus. Patient and donor HLA-DPB1 genotyping was performed by reverse SSO typing and the ImMunoGeneTics/HLA (IMGT/HLA) T-cell epitope matching algorithm was used to determine mismatch status as permissive or non-permissive. Of 78 donor recipient pairs, there were 13 HLA-DPB1 matches, 42 permissive mismatches, and 23 non-permissive mismatches (10 in the graft-vs-host [GvH] direction, 13 in the host-vs-graft [HvG] direction). In a univariate analysis, when matches and permissive mismatches were combined into a group and compared to non-permissive mismatch, there was no significant difference in overall survival (p=0.31), relapse (p=0.79), or neutrophil or platelet engraftment (p=0.23 and p=0.99, respectively). No significant difference was observed in acute graft-vs-host disease (aGVHD; p=0.25) or grade II-IV aGVHD (p=0.62) among the matched, permissive mismatched, and non-permissive mismatched groups. The permissive mismatched group showed an increase in chronic GVHD (cGVHD) over the matched and the non-permissive mismatched groups that neared significance (p=0.052). In univariate analyses, for non-permissive mismatches, the GvH mismatch direction trended toward reduced survival (p=0.26), increased relapse (p=0.13), increased aGVHD (p=0.064), and increased cGVHD (p=0.22). We did not identify any significant differences in survival, relapse, engraftment, or acute or chronic GVHD among the patients with HLA-DPB1 donor matches, permissive mismatches, and non-permissive mismatches, although our study was limited by small sample size. Further work from large databases is necessary to fully understand the impact on clinical outcomes of HLA-DPB1 mismatches.

A systematic investigation of the contribution of genetic variation within the MHC region to HPV seropositivity.

High-risk mucosal types of human papillomavirus (HPV) cause anogenital and oropharyngeal cancers, whereas cutaneous types (e.g. HPV8 and 77) are suspected to be involved in non-melanoma skin cancer. The antibody response to HPVs is a key determinant of protective immunity, but not all infected individuals seroconvert. Genetic variability of the host may have large impact on seroconversion. A previous genome-wide association study (GWAS) has identified a susceptibility locus (rs41270488) for HPV8 seropositivity within the major histocompatibility complex (MHC) region. To further study this locus, we imputed alleles at classical leukocyte antigen (HLA) loci using HLA*IMP:02 with a reference panel from the HapMap Project and the 1958 Birth Cohort, and conducted an integrated analysis among 4811 central European subjects to assess the contribution of classical HLA alleles and gene copy number variation (CNV) at the hypervariable DRB locus within the MHC region to HPV seropositivity at both the individual HPV type level and the phylogenetic species level. Our study provides evidence that the association noted between rs41270488 and HPV8 seropositivity is driven by two independent variants, namely DQB1*0301 [odds ratio (OR) = 1.51, 95% confidence interval (CI) = 1.36-1.68, P = 1.0 × 10(-14)] and DRB1*1101 (OR = 1.89, 95%CI = 1.57-2.28, P = 1.5 × 10(-11)) within the HLA class II region. Additionally, we identified two correlated alleles DRB1*0701 (OR = 1.67, 95%CI = 1.41-1.98, P = 2.6 × 10(-9)) and DQA1*0201 (OR = 1.67, 95%CI = 1.38-1.93, P = 1.7 × 10(-8)), to be associated with HPV77 seropositivity. Comparable results were observed through imputation using SNP2HLA with another reference panel from the Type 1 diabetes Genetics Consortium. This study provides support for an important role of HLA class II alleles in antibody response to HPV infection.

Retinal regeneration with iPS cells ‒ Clinical trials for retinal degenerative disorders

Potential for re-programming cells has become widely accepted as a tool for obtaining transplantation materials. There has been great interest in cell-based therapies, including retinal transplants, because there is a reduced risk of immune rejection. Stem cells have the capacity for self-renewal plus the capacity to generate several differentiated cells. They are derived from many sources including human adult-derived induced pluripotent stem (iPS) cells and have found early application in the context of ocular disease. In results, our established iPS-retinal pigment epithelial (RPE) cells are high-quality RPE cells. iPS cells-derived RPE cells clearly showed polygonal morphology (mostly hexagonal) and contained melanin. Moreover, RPE cells derived from iPS cells had many characteristics of mature RPE cells in vivo, but no characteristics of pluripotent stem cells. Recently, we transplanted RPE cell sheets to treat a patient with wet age-related macular degeneration (September, 2014). In addition, we are now conducting experiments to determine whether allogeneic T cells can recognize iPS-RPE cells from HLA-A, B, DRB1 locus homozygote donors. iPS bank might be useful as allografts in retinal disorders, if the recipient T cells cannot respond to allogeneic RPE cells because of match to some of main HLA antigens.

Polymorphisms of HLA-DRB1, -DQA1 and -DQB1 in inhabitants of Astana, the capital city of Kazakhstan.

BackgroundKazakhstan has been inhabited by different populations, such as the Kazakh, Kyrgyz, Uzbek and others. Here we investigate allelic and haplotypic polymorphisms of human leukocyte antigen (HLA) genes at DRB1, DQA1 and DQB1 loci in the Kazakh ethnic group, and their genetic relationship between world populations.Methodology/Principal FindingsA total of 157 unrelated Kazakh ethnic individuals from Astana were genotyped using sequence based typing (SBT-Method) for HLA-DRB1, -DQA1 and -DQB1 loci. Allele frequencies, neighbor-joining method, and multidimensional scaling analysis have been obtained for comparison with other world populations. Statistical analyses were performed using Arlequin v3.11. Applying the software PAST v. 2.17 the resulting genetic distance matrix was used for a multidimensional scaling analysis (MDS). Respectively 37, 17 and 19 alleles were observed at HLA-DRB1, -DQA1 and -DQB1 loci. The most frequent alleles were HLA-DRB1*07:01 (13.1%), HLA-DQA1*03:01 (13.1%) and HLA-DQB1*03:01 (17.6%). In the observed group of Kazakhs DRB1*07:01-DQA1*02:01-DQB1*02:01 (8.0%) was the most common three loci haplotype. DRB1*10:01-DQB1*05:01 showed the strongest linkage disequilibrium. The Kazakh population shows genetic kinship with the Kazakhs from China, Uyghurs, Mongolians, Todzhinians, Tuvinians and as well as with other Siberians and Asians.Conclusions/SignificanceThe HLA-DRB1, -DQA1and -DQB1 loci are highly polymorphic in the Kazakh population, and this population has the closest relationship with other Asian and Siberian populations.

The Impact of HLA and KIR Ligand Mismatching on Unrelated Allogeneic Hematopoietic Stem Cell Transplantation in Korean Adult Patients

BackgroundThe impact of HLA and KIR ligand mismatching on the outcome of hematopoietic stem cell transplantation (HSCT) remains unclear. Previous reports have identified considerable ethnic differences in the impact of HLA and KIR ligand mismatches, as well as KIR ligand status, on HSCT; however, to date, no data has been acquired in Korean adult patients.MethodsWe investigated the association of high-resolution HLA matching on five loci (HLA-A, -B, -C, -DRB1, and -DQB1), KIR ligand mismatching, and KIR ligand status on the outcome of allogeneic HSCT from unrelated donors in 154 Korean adult patients treated at Seoul National University Hospital.ResultsIn a multivariate analysis, less than 9/10 allelic matches in five HLA loci was an independent risk factor for acute graft-versus-host disease (GVHD) (grade II to IV) (P=0.019, odds ratio [OR]=2.7). In addition, HLA-A allele mismatching was increasingly prevalent in patients with acute GVHD compared to patients without (61.9% vs. 34.5%, P=0.06). For KIR ligand status, the patient and donor combination of both C1/C1 ligands showed better event-free and overall survival than combinations with C2 ligand patients or donors (P=0.048, P=0.034, respectively) by log-rank test.ConclusionsKorean adult transplant patients with less than 9 of 10 HLA allele matches in the HLA-A, -B, -C, -DRB1, and DQB1 loci have a higher likelihood of developing acute GVHD (grade II to IV). Impact of KIR ligand status on clinical outcome should be further studied in a larger patient population.

Clinical Relevance of HLA Supertype Matching after Myeloablative Conditioning 7/8 Unrelated Donor Hematopoietic Cell Transplantation: A CIBMTR Study

The diversity of the HLA class I and II alleles can be simplified by consolidating them into fewer supertype clusters based on functional or predicted structural similarities in epitope binding grooves of HLA molecules. HLA class I and II supertypes have been increasingly studied in association with immune susceptibility to infection and cancer with potential implications for vaccine development. However, the significance of individual allele mismatching within and outside of HLA class I or II supertypes remains unknown in the context of hematopoietic cell transplantation (HCT). We therefore studied the impact of HLA supertype disparities on clinical outcomes of 1934 patients with AML (45%), ALL (31%), CML (14%) or MDS (9%) who underwent 7/8 unrelated donor myeloablative conditioning HCT from 1999 to 2011 and were registered with CIBMTR. Median age at transplant was 35 years (range, 1-70); 53% were males; 81% Caucasian; 56% received peripheral blood grafts; 50% were ABO-mismatched; 36% had in-vivo T-cell depletion; 62% received tacrolimus- and 36% cyclosporine A-based GVHD prophylaxis; 72% male or non-parous female donors; median follow up of survivors was 54 months (3-149). Supertype assignment methods of (1) revised main HLA anchor specificities (Sydney, 2008) and (2) bioinformatics (Doytchinova, 2004-05) were used to categorize single mismatched alleles into 6 HLA-A (A01, A01A03, A01A24, A02, A03, A24), 6 HLA-B (B07, B08, B27, B44, B58, B62), 2 HLA-C (C1, C2), and 5 DRB1 (DR1, DR3, DR4, DR5, DR9) supertypes. Overall survival (OS), disease-free survival (DFS), relapse, treatment-related mortality (TRM), acute graft vs. host disease (aGVHD) and chronic GVHD were compared across matched vs. mismatched HLA-A (265 vs. 429), -B (230 vs. 92), -C (365 vs. 349), and -DRB1 (153 vs. 51) supertypes. We used predetermined α=0.01 for statistical significance as multiple exploratory analyses were conducted by Kaplan-Meier, Gray, and Cox proportional hazard methods. In the multivariable analysis, supertype B-mismatch was associated with increased risk of grade II-IV aGVHD (HR=1.78; 95% CI, 1.23-2.59, p=0.0025), however no difference was found for grade III-IV aGVHD or other clinical outcomes compared to supertype B-matches. Supertype DRB1-mismatch was associated with shorter neutrophil recovery (HR=0.51; 95% CI, 0.36-0.71, p=0.0001), yet a trend toward inferior OS (HR=1.58; 95% CI 1.04-2.38, p=0.037) and higher TRM (HR=1.64; 95% CI, 0.99-2.74, p=0.0565) compared to DRB1 matches within supertypes. There was no increased risk of GVHD with DRB1 supertype mismatch. No associations were observed between HLA-A and -C supertypes or aggregate supertype-matched vs. -mismatched groups for any outcomes. Our analysis demonstrated differential influence of HLA supertype-based allele matching within -B and -DRB1 loci on clinical outcomes after myeloablative 7/8 URD HCT. DisclosuresNo relevant conflicts of interest to declare.

From genome-wide to candidate gene: an investigation of variation at the major histocompatibility complex in common bottlenose dolphins exposed to harmful algal blooms.

The role the major histocompatibility complex (MHC) plays in response to exposure to environmental toxins is relatively poorly understood, particularly in comparison to its well-described role in pathogen immunity. We investigated associations between MHC diversity and resistance to brevetoxins in common bottlenose dolphins (Tursiops truncatus). A previous genome-wide association study investigating an apparent difference in harmful algal bloom (HAB) resistance among dolphin populations in the Gulf of Mexico identified genetic variation associated with survival in close genomic proximity to multiple MHC class II loci. Here, we characterized genetic variation at DQA, DQB, DRA, and DRB loci in dolphins from central-west Florida and the Florida Panhandle, including dolphins that died during HABs and dolphins presumed to have survived HAB exposure. We found that DRB and DQB exhibited patterns of genetic differentiation among geographic regions that differed from neutral microsatellite loci. In addition, genetic differentiation at DRB across multiple pairwise comparisons of live and dead dolphins was greater than differentiation observed at neutral loci. Our findings at these MHC loci did not approach the strength of association with survival previously described for a nearby genetic variant. However, the results provide evidence that selective pressures at the MHC vary among dolphin populations that differ in the frequency of HAB exposure and that the overall composition of DRB variants differs between dolphin survivors and non-survivors of HABs. These results may suggest a potential role of MHC diversity in variable survival of bottlenose dolphins exposed to HABs.

Optimized Cyclophosphamide (CY) Dosing in Combination with Fludarabine, TBI and ATG As Conditioning for Unrelated Donor Bone Marrow Transplantation (BMT) in Severe Aplastic Anemia (SAA): A Phase I/II Study from the Blood and Marrow Transplant Clinical Trials Network (BMT CTN)

▪Objective: To determine toxicity and efficacy of adding fludarabine (FLU) to a standard preparative regimen of low-dose total body irradiation (TBI), anti-thymocyte globulin (ATG) and CY (Deeg J et al, Blood 108:1485, 2006), with de-escalation of the CY dose.Patients and Methods: Between May 2006 and December 2013, the BMT CTN (sponsored by the NHLBI and NCI) conducted a Phase I/II trial of unrelated donor BMT in SAA (BMT CTN 0301; NCT 00326417). Patients were eligible if they were aged < 65 years, with adequate organ function, and an available unrelated marrow donor matched at 7 or 8 of 8 HLA-A, B, C, and DRB1 loci. The trial accrued 97 patients, although analyses are limited to 96; 1 patient withdrew consent prior to BMT. All patients received TBI 200 cGy (day -1), ATG (either thymoglobulin 3 mg/kg IV or ATGAM 30 mg/kg IV daily x 3, days –4 to –2), FLU (30 mg/m2 IV daily x 4, days –5 to –2). The Phase I portion of the trial tested four CY dose levels: 150 mg/kg (days –4 to –2); 100 mg/kg (days –3 to –2); 50 mg/kg (day –2); and 0 mg/kg. The Phase I design allowed enrollment of up to six patients at each CY dose level unless toxicity or graft failure boundaries were crossed. In the Phase II portion, patients were enrolled onto the optimal CY dose level, using adaptive Bayesian criteria to rank desirability of CY doses based on Day 100 outcomes of engraftment and early death. The primary endpoint of the study was determination of the optimal CY dose based on Day 100 assessments of graft failure (primary: absolute neutrophil count <0.5 x 109/L and secondary: sustained decline in absolute neutrophil count to <0.5 x 109/L), major (grade 3 or higher) regimen-related toxicity (RRT) and early death. Early stopping guidelines were based on a composite endpoint of graft failure and treatment-related mortality through day 100.Results: Twenty-one patients accrued to the Phase I portion of the trial and all CY dose levels were tested. CY dose level 0 mg/kg was closed after 3 of the first 3 patients developed secondary graft failure. The Phase II portion of the trial opened with CY dose level 150 mg/kg. However, this dose level was closed for excess toxicity (7 of 14 patients died; organ failure n=4, ARDS n=2 and infection n=1; Tolar et al, Biol Blood Marrow Transplant, 2012). Patients were subsequently assigned to CY dose level 100 mg/kg (CY DL 100; n=41) or CY dose level 50 mg/kg (CY DL 50; n=38), depending on Bayesian assessment of criteria noted above except that the last 20 patients were preferentially assigned to CY DL 50 to balance accrual to the two dose levels. Approximately half of patients were male and 79% Caucasian. The median age was 20.6 years. The median age for CY DL 50 and DL 100 was 24.5 (0.5-65) and 17.6 (1.9-63) years, respectively. The number of transplants with a mismatched (i.e.7/8) donor was 7 (18%) in the CY DL 50 group and 14 (34%) in the CY DL 100 group. All patients reached their Day 100 endpoint. The cumulative incidence of grade 2-4 acute GVHD was comparable between the two CY dose levels: 24% (DL 50) vs 27% (DL 100). All deaths before Day 100 were due to primary or secondary graft failure (CY DL 50; n=1 and CY DL 100; n=2),. Table 1 shows individual Day 100 outcomes for CY DL 50 and CY DL 100. Median follow up is 15 (3-27) and 24 (12-50) months for CY DL 50 and 100, respectively. All surviving patients on CY dose level 100 mg/kg have achieved a minimum follow up of 12 months, as opposed to only 75% of surviving patients on CY dose level 50 mg/kg.Conclusion: Results of the adaptive Bayesian dose selection suggest that the most desirable CY dose is 50 mg/kg followed by CY dose, 100 mg/kg. However, interpretation of data needs to take into account the potential imbalance of donor-recipient HLA disparity in the CY 50 mg/kg dose level. CY dose 150 mg/kg and 0 mg/kg should be avoided.Table 1Day 100 OutcomeCY DL 50 (n=38)CY DL 100 (n=41)Graft Failure, primary and secondary3 (8%)6 (15%)Survival37 (97%)39 (95%)Major RRT (Grade III or higher)4 (11%)9 (22%) DisclosuresNo relevant conflicts of interest to declare.