Sequence-specific Oligonucleotide Typing Research Articles

Human leukocyte antigen association in systemic sclerosis patients: our experience at a tertiary care center in North India.

Systemic sclerosis (SSc) is a chronic multisystem autoimmune rheumatic disease of unknown etiology. Several studies have established that SSc is triggered by a dynamic interplay between genetic factors and environmental stimuli. In the present study, we aimed to study the association of human leukocyte antigen (HLA) with familial and non-familial SSc patients [limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc)] from North India. The HLA-A, B, DRB1, and DQB1 genotyping of 150 (70 lcSSc and 80 dcSSc) adult-onset SSc patients and 150 age-gender-matched healthy controls were performed with sequence-specific oligonucleotide (SSO) typing kits using the luminex platform. HLA typing for HLA class I (A, B, and C) and II (DRB1, DQB1, and DPB1) in five North Indian families consisting of parent-child/sibling pairs affected with SSc or overlap syndrome was performed by Next Generation Sequencing (NGS) with Illumina MiniSeq. Among the non-familial SSc patients, HLA- DRB1*11 (P = 0.001, OR: 2.38, P c = 0.01) was identified as a risk allele, and DRB1*12 (P = .0001, OR: 0.00, P c = 0.001) as a protective allele. There was no statistical association found with HLA-DQB1*. Also, no significant association was observed between HLA antigens and different clinical subsets (lcSSc and dcSSc) of SSc. Two cases of familial SSc patients had the DRB1*11 allele. The DRB1*12 allele was absent in all the familial SSc patients. HLA DRB1*11 (risk allele) and DRB1*12 (protective allele) were found to be strongly associated with non-familial SSc patients and partially explain the disease's familial clustering, supporting the susceptible genetic background theory for SSc development. The study also indicates the HLA allele as a common genetic risk factor in distinct autoimmune diseases contributing to overlap syndrome or polyautoimmunity.

Development of cost-effective and fast KIR genotyping by multiplex PCR-SSP.

Killer-cell immunoglobulin-like receptors (KIR) control natural killer (NK) cell functions by recognizing HLA molecules and modulating the activity of NK cells. The KIR gene cluster contains polymorphic and highly homologous genes. Diversity of the KIR region is achieved through differences in gene content, allelic polymorphism, and gene copy number, which result in unrelated individuals having different KIR genotypes and individualized immune responses that are relevant to multiple aspects of human health and disease. Therefore, KIR genotyping is increasingly used in epidemiological studies. Here, we developed multiplex polymerase chain reaction with sequence-specific primers (PCR-SSP) to compensate for the shortcomings of the conventional PCR-SSP method, which is most commonly used for KIR analysis. Multiplex PCR-SSP method involves six multiplex reactions that detect 16 KIR genes and distinguish variant types of some KIR genes by adding two reactions. The assay was evaluated in a blind survey using a panel of 40 reference DNA standards from the UCLA KIR Exchange Program. The results are 100% concordant with the genotype determined using Luminex-based reverse sequence-specific oligonucleotide typing systems. Additionally, we investigated the currently known 16 KIR genes and their common variants in 120 unrelated Korean individuals. The results were consistent with the KIR genotype previously reported by Hwang et al. This multiplex PCR-SSP is an efficient method for analyzing KIR genotypes in both small- and large-scale studies with minimal labor, reagents, and DNA. Furthermore, by providing a better definition of KIR polymorphisms it can contribute to developments in immunogenetics.

Association between Human Leukocyte Antigen (HLA) DQB1*06 and HLA DQB1*03 and adverse outcomes in a group of critically ill patients with COVID-19 in Tunisia: a cross-sectional study.

Human Leukocyte Antigen (HLA) system is a highly polymorphic genetic system associated with the prognosis of several infectious diseases. The aim of this study is to investigate the association of HLA polymorphism with the outcome of coronavirus disease 2019 (COVID-19) in Tunisian critically ill patients. this retrospective cross-sectional study included 42 consecutive patients hospitalized in intensive care unit (ICU) for COVID-19 in March 2021. Genotyping of HLA loci was performed by LABType™ sequence-specific oligonucleotide (SSO) typing kits (One lambda Inc, USA). Statistical analyses were performed using Statistical Package for Social Sciences (SPSS®) version 23.0. A p-value <0.05 was considered significant. Multivariable regression analysis was performed for the association between HLA polymorphism with adverse outcomes with adjustment for potential confounders such as age, sex, co-morbidities and blood type. patients included in our study had a mean age of 64.5 ± 11.5 (34-83) years and were mainly men (64.3%; (n=27)). The most common cardiovascular risk factors were obesity (61.9%; (n=26)) and hypertension (26.2%; (n=11)). Thirty-two patients died (76.2%). Eleven patients (26.2%) required intubation during hospitalization. We found that HLA DQB1*06 allele was significantly associated with protection against mortality aOR: 0.066, 95% CI 0.005-0.821; p = 0.035. HLA DQB1*03 allele was significantly associated with protection against intubation aOR: 0.151, 95% CI 0.023-0.976; p = 0.047. it was found that there are 2 protective HLA alleles against COVID-19 severity and mortality in critically ill patients. This could allow focusing on people genetically predisposed to develop severe forms of COVID-19.

KIR- Ligand Interactions in Hypertensive Disorders in Pregnancy.

HypothesisThe activity of natural killer (NK) cells is considered an important factor for the tolerance of the fetus during pregnancy. The complications of pregnancy, such as hypertensive disorders (HDP), may be therefore associated with this immune compartment.MethodsThe current study included 41 pregnant women diagnosed with HDPs (Gestational Hypertension; GH or Preeclampsia; PE) and 21 healthy women. All the patients were under continuous obstetric care during the pregnancy and labour. The number of mother-child mismatches within killer immunoglobulin-like receptors (KIRs), their ligands [MM], and missing KIR ligands [MSLs] was assessed. KIRs and their ligands were assessed with Next Generation Sequencing (NGS) and Polymerase Chain Reaction Sequence-Specific Oligonucleotide (PCR-SSO) typing. The subsets of NK cells were assessed with multicolor flow cytometry and correlated to the number of MSLs.ResultsThe number of MSLs was significantly higher in HDP patients when compared to healthy non-complicated pregnancy patients. Some MSLs, such as those with 2DS2 activating KIR, were present only in HDP patients. The percentage of CD56+CD16-CD94+ NK cells and CD56+CD16-CD279+ NK cells correlated with the number of MSLs with inhibiting KIRs only in healthy patients. In HDP patients, there was a correlation between the percentage of CD56-CD16+CD69+ NK cells and the number of MSLs with inhibiting and activating KIRs. As compared to the healthy group, the percentage of CD56+CD16-CD279+ NK cells and CD56-CD16+CD279+ NK cells were lower in HDP patients. HDP patients were also characterized by a higher percentage of CD56+CD16+perforin+ NK cells than their healthy counterparts.ConclusionsPatients with HDP were characterized by a higher number of MSLs within the KIRs receptors. It seemed that the number of MSLs in the healthy group was balanced by various receptors, such as CD94 or inhibitory CD279, expressed on NK cells. Conversely, in HDP patients the number of MSLs was associated with the activation detected as the increased level of CD69+ NK cells.

The Accuracy of Sequence-Specific Oligonucleotide and Real-Time Polymerase Chain Reaction HLA Typing in Determining the Presence of Pre-Transplant Donor-Specific Anti-HLA Antibodies and Total Eplet Mismatches for Deceased Donor Kidney Transplantation

High resolution human leukocyte antigen (HLA) typing is important in establishing eplet compatibility and the specificity of donor-specific anti-HLA antibodies (DSA). In deceased donor kidney transplantation, high resolution donor HLA typing may not be immediately available, leading to inaccuracies during the organ allocation process. We aimed to determine the concordance and agreement of HLA-Class I and II eplet mismatches calculated using population frequency based allelic haplotype association (linkage disequilibrium, LD) from sequence-specific oligonucleotide (SSO) and real-time polymerase chain reaction (rtPCR) donor HLA typing (available at time of donor kidney allocation) compared to high-resolution Next Generation Sequencing (NGS) donor typing. NGS high resolution HLA typing were available for all recipients prior to donor kidney allocation. A cohort of 94 deceased donor-recipient pairs from a single Western Australian center were included (77 individual donors typed, 55 local and 22 interstate). The number of class I (HLA-A+B+C) and class II (HLA-DRB1+DRB3/4/5+DQB1+DQA1+DPB1+DPA1) eplet mismatches were calculated using HLAMatchmaker, comparing LD- and NGS-HLA typing. The accuracy in assigning pre-transplant DSA was compared between methods. The concordance correlation coefficient (95%CI) for HLA-class I and II eplet mismatches were 0.994 (0.992 to 0.996) and 0.991 (0.986 to 0.993), respectively. The 95% limits of agreement for class I were -1.3 (-1.6 to -1.1) to 1.4 (1.2 to 1.7) and -4.8 (-5.7 to -3.9) to 5.0 (4.1 to 5.9) for Class II. Disagreement between the two methods were present for 11 and 37 of the Class I and II donor/recipient pairs. Of which, 5 had a difference of ≥5 class II eplet mismatches. There were 34 (36%) recipients with potential pre-transplant DSA, of which 8 (24% of recipients with DSA) had indeterminate and ultimately false positive DSA assigned by donor LD-typing. While the concordance between NGS- and LD-typing was high, the limits of agreement suggest meaningful differences between these two techniques. The inaccurate assignment of DSA from donor LD-typing may result in associated HLA being considered unacceptable mismatches, inappropriately precluding candidates’ access to transplantation. Accurate imputation of two-field HLA alleles based on LD from SSO and rtPCR HLA typing remains a substantial challenge in clinical practice in-lieu of widely available, rapid, high-resolution methods.

Association of HLA-DRB1 and -DQB1 Alleles with Susceptibility to IgA Nephropathy in Korean Patients.

BackgroundAssociations between IgA nephropathy (IgAN) and HLA-DRB1 and -DQB1 alleles have been reported in several ethnic groups. We investigated the association of HLA-DRB1 and -DQB1 alleles with the predisposition for IgAN and disease progression to end-stage kidney disease (ESKD) in Korean patients.MethodsWe analyzed HLA-DRB1 and -DQB1 genotypes in 399 IgAN patients between January 2000 and January 2019 using a LIFECODES sequence-specific oligonucleotide (SSO) typing kit (Immucor, Stamford, CT, USA) or a LABType SSO Typing Test (One Lambda, Canoga Park, CA, USA). Alleles with a significant difference in two-digit resolution were further analyzed using in-house sequence-based typing and sequence-specific primer PCR. As controls, 613 healthy hematopoietic stem cell donors were included. Kidney survival was analyzed in 281 IgAN patients with available clinical and laboratory data using Cox regression analysis. Where needed, P-values were adjusted using Bonferroni correction.ResultsThe allele frequencies of HLA-DRB1*0405 (corrected P [Pc]<0.001), -DQB1*0401 (Pc=0.048), and -DQB1*0302 (Pc=0.021) were significantly higher in IgAN patients than in controls, whereas those of HLA-DRB1*0701, -DRB1*1501, -DQB1*0202, and -DQB1*0602 (Pc<0.001 for all) were significantly lower in IgAN patients than in controls. The allele frequency of HLA-DQB1*0503 (Pc=0.016) was significantly lower in the ESKD group than in the non-ESKD group; however, there was no significant difference for ESKD progression between these groups.ConclusionsWe report novel associations of HLA-DRB1*1501, DQB1*0202, -DQB1*0302, and -DQB1*0401 with IgAN. Further studies of HLA alleles associated with IgAN progression in a larger cohort and in various ethnic groups are needed.

Association between human leukocyte antigen gene polymorphisms and multiple EPIYA-C repeats in gastrointestinal disorders

BACKGROUNDPolymorphisms of human leukocyte antigen (HLA) genes are suggested to increase the risk of gastric cancer (GC).AIMTo investigate the HLA allele frequencies of patients with GC relative to a control group in terms of CagA+ multiple (≥ 2) EPIYA-C repeats.METHODSThe patient group comprised 94 patients [44 GC and 50 duodenal ulcer (DU) patients], and the control group comprised 86 individuals [(50 non-ulcer dyspepsia patients and 36 people with asymptomatic Helicobacter pylori (H. pylori)]. Polymerase chain reaction was performed for the amplification of the H. pylori cagA gene and typing of EPIYA motifs. HLA sequence-specific oligonucleotide (SSO) typing was performed using Lifecodes SSO typing kits (HLA-A, HLA-B HLA-C, HLA-DRB1, and HLA-DQA1-B1 kits).RESULTSThe comparison of GC cases in terms of CagA+ multiple (≥ 2) EPIYA-C repeats showed that only the HLA-DQB1*06 allele [odds ratio (OR): 0.37, P = 0.036] was significantly lower, but significance was lost after correction (Pc = 0.1845). The HLA-DQA1*01 allele had a high ratio in GC cases with multiple EPIYA-C repeats, but this was not significant in the univariate analysis. We compared allele frequencies in the DU cases alone and in GC and DU cases together using the same criterion, and none of the HLA alleles were significantly associated with GC or DU. Also, none of the alleles were detected as independent risk factors after the multivariate analysis. On the other hand, in a multivariate logistic regression with no discriminative criterion, HLA-DQA1*01 (OR = 1.848), HLA-DQB1*06 (OR = 1.821) and HLA-A*02 (OR = 1.579) alleles were detected as independent risk factors for GC and DU.CONCLUSIONNone of the HLA alleles were detected as independent risk factors in terms of CagA+ multiple EPIYA-C repeats. However, HLA-DQA1*01, HLA-DQB1*0601, and HLA-A*2 were independent risk factors with no criterion in the multivariate analysis. We suggest that the association of these alleles with gastric malignancies is not specifically related to cagA and multiple EPIYA C repeats.

Predictors of drug survival of biologic therapies in psoriasis patients

Introduction We aimed to investigate the clinical, immunological, and genetic factors affecting the response to anti-TNFα (tumor necrosis factor-α) and interleukin-12/23 therapies and drug survivals. Methods A total of 180 patients were divided into two groups: 89 patients who used at least two biologic agents, with the initial biologic agent used less than 12 months (group A), and 91 biologic-naive patients who have been receiving a single biologic agent for more than 12 months (group B). ELISA (enzyme-linked immunosorbent assay) was used to analyze anti-drug antibodies (ADAs) in blood samples. Clinical data of the patients were retrospectively analyzed. HLA-SSO (sequence-specific oligonucleotide) Typing Kits were used for HLA-C typing. IBM SPSS v.21 was used for statistical analysis. Results: Infliximab had the longest drug survival as the first biologic agent in group A (p = .015). Etanercept had the lowest ADA count compared to the other anti-TNF agents (p = .001). HLA-Cw6 negativity, late-onset psoriasis, smoking and alcohol use were determined to be risk factors for treatment failure in group A. HLA-Cw6 was found to be associated with type I psoriasis (p = .000). Conclusions Although our study is retrospective of a relatively low number of patients, this is a preliminary study focusing on two different patient populations based on therapy response.

P013 Steps leading to discovery of a rare allele

Sanger sequencing-based typing (SBT, Life Technologies), and LABType Sequence specific oligonucleotide (SSO) Typing (One Lambda) are performed on all bone marrow/stem cell transplant recipients and prospective donors at Mayo Clinic, Rochester. SSO performed on an 11 yr-old African American male with Hemophagocytic Lymphohistiocytosis needing a stem cell transplant and his full-sibling donor, resulted in C*04/C*07 with group 1 results showing common allele possibilities. SBT of exons 2, 3, and 4 resulted in either a rare C*04 allele, a rare C*07 allele, or both C*04 and C*07 being rare alleles. (Fig. 1a). At base pair 862 in exon 4, a heterozygous A/G result was seen. At this base the G result was consistent with a C*07:18 while the A suggested C*04:165. (Fig. 1b). This nucleotide exchange from G → A in codon 264 resulted in an amino acid change from Glutamic Acid to Lysine GAG → AAG. Ambisolv Sequence Specific Primers (SSP, One Lambda) were performed to help differentiate the common alleles C*04:09 N, C*04:82, C*07:01, C*07:06, and C*07:18. Except for the C*07:18, all were negative and led to an initial interpretation of C*04:165 and C*07:18. Olerup C*04 and C*07 SSP trays were set up with C*04:01/C*07:18 results obtained. Included in the C*04 SSP tray was C*04:165 with negative results. To further confirm the presence of C*07:18, exons 5 and 6 were sequenced by SBT where results at position 1043 showed a T/C. (Fig. 1b). After SSO typing a maternal half-sibling showing a haplotype match at class I, SBT was performed showing a C*04:01 homozygous result. (Fig. 1c). To help resolve the typing, Next Generation Sequencing (NGS) using Omixon Holotype HLA was performed. This led to a conclusion of C*04:01/C*07:18 novel allele (Fig. 1d). The allele has been newly discovered and named C*07:607 in the IPD-IMGT/HLA Database. It was originally found in a Black East African female. NGS phasing proved extremely valuable in identifying the nucleotide change from G→A at position 862 (codon 264) belonging to the C*07 and not the C*04.

Accelerated Progression to Type 1 Diabetes in the Presence of HLA-A*24 and -B*18 Is Restricted to Multiple Islet Autoantibody-Positive Individuals With Distinct HLA-DQ and Autoantibody Risk Profiles.

We investigated the effect of HLA class I risk alleles on disease progression in various phases of subclinical islet autoimmunity in first-degree relatives of patients with type 1 diabetes. A registry-based group of siblings/offspring (aged 0-39 years) was monitored from single- to multiple-autoantibody positivity (n = 267) and from multiple-autoantibody positivity to clinical onset (n = 252) according to HLA-DQ, -A*24, -B*18, and -B*39 status. Genetic markers were determined by PCR sequence-specific oligotyping. Unlike HLA-B*18 or -B*39, HLA-A*24 was associated with delayed progression from single- to multiple-autoantibody positivity (P = 0.009) but not to type 1 diabetes. This occurred independently from older age (P < 0.001) and absence of HLA-DQ2/DQ8 or -DQ8 (P < 0.001 and P = 0.003, respectively), and only in the presence of GAD autoantibodies. In contrast, HLA-A*24 was associated with accelerated progression from multiple-autoantibody positivity to clinical onset (P = 0.006), but its effects were restricted to HLA-DQ8+ relatives with IA-2 or zinc transporter 8 autoantibodies (P = 0.002). HLA-B*18, but not -B*39, was also associated with more rapid progression, but only in HLA-DQ2 carriers with double positivity for GAD and insulin autoantibodies (P = 0.004). HLA-A*24 predisposes to a delayed antigen spreading of humoral autoimmunity, whereas HLA-A*24 and -B*18 are associated with accelerated progression of advanced subclinical autoimmunity in distinct risk groups. The relation of these alleles to the underlying disease process requires further investigation. Their typing should be relevant for the preparation and interpretation of observational and interventional studies in asymptomatic type 1 diabetes.

P201 Erroneous HLA-B allele typing by sequencing-based typing method that utilizes combinations of Sanger sequencing and rSSO typing

Allele-level HLA matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplantation (HSCT). Sanger sequencing has been the gold standard for allele-level HLA typing since 1996. The typing strategy typically employs the sequencing of exons 2 and 3 for HLA Class I and exon 2 for HLA Class II. These exons encode the antigen recognition domains and sequencing only these exons are considered to be clinically relevant for HSCT. Sanger sequencing results significant number of ambiguities in HLA typing due to partial sequencing of limited exons, inability to phase heterozygous alleles, and growing number of new HLA alleles. Additional typing methods are required to resolve phasing and ambiguity problems. Next-generation sequencing (NGS) permits sequencing of the entire HLA gene and generates unambiguous and phased-resolved HLA typing results up to four field levels. Here we describe an erroneous HLA-B allele typing by Sanger sequencing. Sanger sequencing (Genome Diagnostics B.V.) of an unrelated NMDP donor showed the following possible HLA types: B∗15:01:01,18:14 or B∗15:07:01,18:01:01 or B∗15:07:01,18:17 N or B∗15:07:03,18:01:10. The B∗15:01:01, B∗15:07:01, B∗18:01:01, and B∗18:14 are common and well-documented alleles. The high-definition reverse sequence-specific oligonucleotide (rSSO) typing (One Lambda) ruled out all possible types except B∗15:01:01,18:14. The rSSO bead for B∗15:07:01 showed reactivity below the specified cutoff. We performed NGS typing on this sample, which enabled phasing of polymorphic positions 363 and 485 in exon 3 resulted unambiguous B∗15:07:01,18:01:01 typing. In summary, the strategy of Sanger sequencing plus rSSO typing resulted in incorrect HLA-B assignment for the NMDP donor, which in turn caused the exclusion of matched donor. The incorrect HLA allele assignment can be detrimental to HSCT recipient survival. NGS provides the capability to phase polymorphisms thereby eliminating most ambiguities and provides high-resolution HLA typing without reflexive, time-consuming and expensive additional testing, and therefore NGS is the new gold standard for allele-level HLA typing.

HLA-A, HLA-B, HLA-DRB1 allele and haplotype frequencies of 14,529 Chinese Han bone marrow donors living in Dalian, China.

We investigated the allele and haplotype frequencies of HLA-A, HLA-B and HLA-DRB1 loci in Dalian Chinese Han population using blood samples of unrelated marrow donors who live in Dalian. The genetic relationship between Dalian and different regions worldwide was further explored based on HLA status of different populations. A total of 14,529 samples were genotyped at 2-digit level only by sequence-specific oligonucleotide and sequence-based typing methods. Allele frequencies of HLA-A, HLA-B and HLA-DRB1 were calculated by the direct counting method. Haplotype frequencies and linkage disequilibrium (LD) values were calculated by the maximum likelihood method. F(ST) values were calculated by allele frequency data of each locus. Phylogeny tree of Nei's DA genetic distances was constructed by the UPGMA method. HLA-A*02 was the most frequent allele at HLA-A locus followed by A*11 and A*24. Alleles at HLA-B locus ranked in decreasing order by frequency were B*40, B*15 and B*13. The three highest frequency alleles were DRB1*15, DRB1*09 and DRB1*12 at HLA-DRB1 locus. A*30-B*13-DRB1*07 was the most frequent three-locus haplotype. For the population relationships, Dalian had a relative close genetic relationship with Liaoning and Yantai-Weihai and a relative distant genetic relationship with Australia. The information obtained in this study may provide useful information for anthropological studies, for disease-association studies and helping bone marrow transplantation patients to search HLA-matched donors.

Resolution of HLA-A*68:11N allele by One Lambda labtype® reverse sequence specific oligonucleotide typing kit

Aim Resolution of the ambiguous combination of HLA-A*68:01:02:01/68:11N allele in HSCT patient and donor pairs is now mandatory by NMDP Policy. Laboratories are required to test for non-expressed alleles when they exist as a possibility within the assigned G group. In our laboratory this occurs in the case of A*68:01:02:01 which gives an ambiguous A*68:01/68:11N result by the Sanger sequencing-method. Method One Lambda® HD rSSO kits have developed several probes to identify rare or uncommon alleles with no positive quality control due to the very uncommon/rare nature of these alleles. Therefore, it is difficult to assess whether we can actually identify those specificities with confidence. The Fred Hutchinson Cancer Research Center’s Research Cell Bank made available to us two DNA samples from the 13th IHWG workshop characterized as having A*68:11N allele (9418 and 9419). In addition, several samples with A*68:01:02:01 and/or A*68:02G were included to ensure that the definitive bead #94 for the A*68:11N allele would not go false positive for other expressed A*68 alleles. Results Using One Lambda’s RSSOH1A® lot 008 HLA typing kit we tested IHWG workshop samples 9418 and 9419. Sample 9418 tested as A*03:MJMJ and A*68:11N. Sample 9419 typed as A*24:02G and A*68:01:02:01/68:11N/68:33. Inspection of sample 9419 showed that A*68:01:02:01 and 68:33 were associated in combination with an A*24:36N (a highly unlikely event), thus ruling out the possibility of A*68:01:02:01 or 68:33. Bead #94 was strongly reactive with both samples, indicating the presence of the HLA-A*68:11N allele. Although bead #94 should be positive with other rare HLA-A alleles, the bead should not be positive with the ambiguous combination found in Sanger sequencing. Conclusion One Lambda’s RSSOH1A® test kit can positively identify the A*68:11N allele, which in combination with Sanger sequencing can resolve the ambiguous A*68:01:02:01/68:11N result.

HLA-DQ Barrier

Panel-reactive antibody (PRA) testing provides assessment of the breadth of sensitization a patient might have against human leukocyte antigen (HLA) antigens. The evolution of calculated PRA (cPRA) reflects the commitment of the transplant community to increase accessibility and promote equity to all patients awaiting kidney transplantation. Recent data from our center and others, however, suggested that a significant diversity of HLA-DQ antigens is not captured, which may lead to inequity in allocating cPRA points. HLA-DRB1-DQA1-DQB1 typing of 2182 individuals was evaluated for this study using Luminex-based sequence-specific oligonucleotide typing. A total of 3182 haplotypes were confirmed to have the level of resolution required for this study. The diversity of HLA-DQαβ alleles is greater than what is apparent using the serologic equivalents. The distribution of these alleles within a serologic group varies, with some alleles being more frequent than others; therefore, their representation within the current cPRA system is inaccurate. Three informative examples are given. Haplotypes of DR antigens with DQαβ alleles did not always follow the common published linkage disequilibrium, especially in populations where there is greater genetic diversity. The current cPRA system does not take into account the distribution of molecular equivalents within DQ serologic specificities. This can result is inequitable allocation of sensitization points and disadvantaging the more sensitized patients. To ameliorate this situation, the United Network for Organ Sharing system should allow inputting HLA-DQαβ alleles both for donor typing and as antibody specificities, which will lead to better representation of unacceptable DQ alleles and improve organ allocation equity.

KIR Gene Repertoire and Hodgkin Lymphoma In Children and Adolescents: A Children's Oncology Group Study

An infectious origin for pediatric Hodgkin lymphoma (HL) has long been suspected and Epstein-Barr virus (EBV) has been detected in ∼50% of tumors. Because EBV infections are ubiquitous, yet only a minute fraction of those infected develops HL, it follows that individual differences in the innate immune system response, including the identification and elimination of virus-infected and tumor cells by natural killer (NK) cells, may contribute to HL susceptibility. NK cell effector responses are modulated by the surface expression of various killer cell immunoglobulin-like receptors (KIR) and their binding to human leukocyte antigen (HLA) class I ligands on target cells. Importantly, the specific KIRs expressed on a given NK cell surface are determined in part by the highly polymorphic KIR genes on chromosome 19q13.4. There is considerable interindividual variability in the type and number of these genes, however, two regions of strong linkage disequilibrium have been identified, defining the centromeric and telomeric haplotype segments, which are flanked on each end by highly conserved genes and joined by a recombination hotspot. Accordingly, the haplotypes for a striking majority (≥93%) of individuals examined to date can be formed combinatorially from 3 common centromeric and 2 telomeric motifs.KIR gene content has been associated with pregnancy outcomes, response to infection, susceptibility to autoimmune disease, and malignancy. Few epidemiologic studies examining KIR genotype and lymphoid neoplasms (LN) have been conducted in children and adults. Generally, these studies have reported inverse associations between presence of activating KIR genes and susceptibility to LN. Given the strong likelihood for a viral etiology and the known correlations with HLA type, we performed for the first time a case-control study of the association between KIR gene content and pediatric/adolescent HL using archived samples.Cases (n=171) diagnosed with HL in 1989-2003 at 3-17 years of age at North American Children's Oncology Group institutions and healthy adult controls (n=332) provided peripheral blood or buccal cell samples. Presence/absence of KIR genes (KIR2DS2, KIR2DL2/2DL3, KIR2DL5, KIR2DS3, KIR2DS5, KIR2DL1, KIR3DS1/3DL1, KIR2DS1, KIR2DS4) was determined using a Luminex DNA-based sequence-specific oligonucleotide (SSO) typing methodology. Each individual's repertoire of genes was then classified into centromeric and telomeric KIR haplotype groups. Case haplotypes were compared to those of controls via unconditional logistic regression, resulting in odds ratios (ORs) and 95% confidence intervals (CIs) for the presence of one or two copies of a B haplotype (A/B and B/B) versus no copies (A/A), where B haplotypes encompass greater numbers of activating KIR. Associations were examined for HL overall, for the nodular sclerosis histologic subtype, and for the 3-9 and 10-17 year age groups.The presence of two centromeric B haplotype motifs was associated with development of pediatric and adolescent HL (OR=1.92, 95% CI: 1.12-3.26), but there was no evidence for an association with telomeric haplotypes. Similar results were observed for nodular sclerosis HL (OR=2.13, 95% CI: 1.02-4.43) and for the younger and older age groups (OR=1.80, 95% CI: 0.69-4.66; OR=1.76, 95% CI: 0.91-3.38), respectively.Our results suggest that activating centromeric KIR may play a role in increasing susceptibility to pediatric and adolescent HL. Although these findings differ from those of prior studies of adult HL and other LN, activating KIR have been positively associated with other virally-driven malignancies, such as nasopharyngeal and cervical carcinomas; underlying mechanisms have yet to be discovered. Examination of compound KIR-cognate HLA ligand genotypes is in progress; additional results will be presented. Disclosures:No relevant conflicts of interest to declare.

138-P: HLA TYPING AMBIGUITY OF SEQUENCE-SPECIFIC OLIGONUCLEOTIDE TYPING PROTOCOLS

Using Shannon’s entropy, we objectively measured the HLA typing ambiguity of various commercial Sequence-Specific Oligonucleotide (SSO) protocols (groups of SSO kits used in tandem). We previously compared the HLA typing ambiguity obtained by serology, allele family level DNA-based typing, SSO, and single-pass sequence based typing (SBT). However, commercial companies have made significant advances in SSO technology in recent years, and those results only evaluated early generic versions of SSO kits. For several US populations, and for 5 HLA loci relevant to transplantation (HLA-A, -B, -C, -DRB1, -DQB1), we identify a set of commonly used SSO protocols. We use population-level haplotype frequency data to generate a cohort of simulated subjects, followed by 5-locus genotype imputation to generate a list of genotypes that would result from SSO typing each simulated subject. The imputation step computes the relative likelihood of each genotype, which is then used for entropy calculation. Distribution of entropies across populations and for each locus is generally similar, while the magnitudes differ. For example, entropy for locus HLA-A in African American population reaches 0.19, while the maximum entropy in Caucasians is 0.055. We generally observed that protocols comprised of newer kits had lower ambiguity across all populations. In certain populations, some newer kits had higher entropy than older ones. This may indicate those kits were designed to detect alleles uncommonly found in those populations, but resolving ambiguity in other populations. Ambiguity in SSO typing kits has reduced substantially since their introduction in the mid-1990s. Results also indicate a European focus toward selection of additional probes, which is suboptimal for typing diverse populations. We hope to use Shannon’s entropy next to evaluate group-specific sequence primer strategies with SBT. These results will guide selection of HLA typing methods by researchers and laboratories.

87-P: HAPLOTYPE ASSOCIATION OF HLA-DQB1∗03:19 AND HLA-DR11 IN SAUDI ARABIAN POPULATION

The Human Leukocyte Antigen-DQB1∗03:19 allele was identified by SSO and sequencing-based typing in prospective solid organ and bone marrow transplant recipients and donors in Kingdom of Saudi Arabia. HLA-DQB1∗03:19 is identical to DQB1∗03:01except for a single nucleotide substitution at position 554 (C/T) in Exon 2, which results in threonine to isoleucine amino acid change. Medium resolution genotyping for HLA-DRB1 and -DQB1 was performed by Luminex-based sequence-specific oligonucleotide (SSO) method. Sequencing based typing (SBT) was performed for high resolution HLA-DQB1 genotyping by Atria Genetics HLA SBT kits. The fluorochrome-labeled DNA fragments were electrophorased by capillary electrophoresis on an ABI 3130 Genetic Analyzer. The sequence analysis was performed using ASSIGN 3.6 software (Conexio Genomics). HLA-DQB1∗03:19 allele was observed frequently in Saudi Arabian deceased donors. Majority of HLA-DQB1∗03:19 positive samples (∼72%) also carried DR11 allele. Based on the known HLA linkage disequilibrium, our data suggest that the haplotype DR11-DQB1∗03:19 is frequent in Saudi Arabian populations. Our data suggests that DQB1∗03:19 was frequently observed in Saudi Arabian deceased donors and majority of time it occurs as DR11-DQB1∗03:19 haplotype.

Novel method for next generation sequencing of KIR by Roche 454 (P3383)

Abstract Natural Killer cells are regulated by the polymorphic Killer Ig-like Receptor family. KIR genotype and expression has been associated with HIV-1 disease progression. We developed a high-throughput method to characterize both DNA genotype and RNA expression of KIR using a novel 454 sequencing method (Roche), for study of HIV and innate immunity. This method uses a 299bp and 395bp amplicon to distinguish and assign long - and short-tailed KIR. These amplicons, from motifs common to 3’ UTR of known KIR genes, amplify both KIR DNA and mRNA. We developed a custom bioinformatics pipeline to analyze 454 sequence reads and it compares each sequence (average 3000 reads per specimen) to a reference alignment of KIR genes via the BLAT algorithm. Using this method, we characterized KIR in a cohort of 700 treatment-naïve adults in early HIV-1 disease. Initial results indicate that our KIR DNA 454 sequencing data agree with KIR DNA genotyping results from mass spectrometry-based typing as well as sequence-specific oligonucleotide typing. KIR3DL1 expression on NK cells by flow cytometry shows 80% agreement with KIR mRNA expression from PBMCs by 454. The use of mRNA- and DNA-based next generation sequencing for human immune receptor genes, such as KIR, will accelerate genetic association and functional genetic studies of diseases of the immune system.

Oriental HLA‐A*11:90 detected in a Taiwanese cord blood sample and the haplotype in association with A*11:90 allele

We report here an HLA-A allele, A*11:90, found in a Taiwanese cord blood sample using DNA sequence-based typing (SBT) protocol after observing an anomalous reaction pattern in a sequence-specific oligonucleotide (SSO) typing exercise. The sequence of A*11:90 is identical to A*11:01:01, the most predominant A*11 variant in Taiwanese, in exon 2 but differs from A*11:01:01 in exon 3 by two nucleotide substitutions at codon 163 (c.487C>G and c.488G>A), resulting R163E. In comparison with the sequence of A*11:02:01, the second most predominant subtype of A*11 in Taiwanese A*11:90 has one nucleotide difference at codon 19 (c.55A>G) in exon 2 resulting K19E and two nucleotides variations at codon 163 (c.487C>G and c.488G>A) in exon 3 resulting R163E. HLA-A*11:90-B*40:02-DRB1*11:01 is the deduced probable HLA haplotype in association with A*11:90. The generation of A*11:90 is thought to involve a DNA recombination event between alleles A*11:01:01 and A*80:01 where A*80:01 donated a fragment of the DNA sequence (from n.t. 487 to n.t. 497) to the recipient sequence of A*11:01:01.

A novel HLA-DRB1 allele, DRB1*08:36, identified by sequence-based typing

A novel human leukocyte antigen (HLA)-DRB1 allele, DRB1*08:36, has been identified during routine sequence-specific oligonucleotide (SSO) typing and sequence-based typing (SBT) in a sample from a registered donor of Chinese Marrow Donor Program. The DRB1*08:36 allele differs from the closest related DRB1*08:03:02 allele by one nucleotide substitution in exon 2, at position 181 which results in an amino acid replacement at codon 32. Tyr is exchanged for His in the novel allele. These findings add a novel member to the HLA-DRB1*08 family.