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The DRB4*01:03:01:02N null allele is predominantly associated with the HLA‐DRB1*07–DQB1*03:03 haplotype. Several recent reports demonstrated a less frequent association of DRB4*01:03:01:02N with DRB1*04 carrying haplotypes in different populations. The ability to identify the presence of null alleles is extremely important in the setting of both solid organ transplants and hematopoietic cell transplants, and characterization of haplotypes carrying null alleles is crucial. In this paper, we report for the first time, an association of DRB4*01:03:01:02N null allele with a DRB1*04:07–DQB1*03:02 haplotype. [ABSTRACT FROM AUTHOR]

Abstract: Identifying an HLA-matched sibling donor for hematopoietic stem cell transplantation (HSCT) is time-consuming and expensive, and often limited by reimbursement caps imposed by insurance providers. To improve the effectiveness and efficiency of screening for HLA-matched siblings, we developed an assay for determining HLA identity using a panel of nine informative short tandem repeat (STR) loci located throughout the HLA complex. The STR panel was assessed for accuracy in identifying HLA-matched siblings in 88 family workups comprising a total of 132 related donor and recipient typing comparisons. All sibling pairs with identical STR alleles were also HLA identical. Of the 48 pairs mismatched at one or more STR alleles, all were genotypically HLA non-identical at one or more loci. The sensitivity and specificity of STR analysis for identifying HLA-matched siblings were 91% and 100%, respectively. Three false negatives occurred due to an STR mutation or possible HLA-DPB1/DQB1 recombination. Additionally, STR genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, due to extensive homozygosity or shared parental haplotypes. The HLA STR assay is a reliable and rapid test that can be used to inexpensively screen potential sibling donors for HLA identity. [Copyright &y& Elsevier]

Abstract: Solid phase antibody assays are increasingly used to provide quantitative measures of donor-specific HLA antibodies for assessment of pretransplant risk, although cell-based crossmatches continue to serve as gold standards for determination of donor HLA antibody strength. This study determined the ability of HLA antibody solid phase assays to predict the strength of cell-based flow cytometric (FC) and complement-dependent cytotoxicity (CDC) crossmatches. Eighty-two recipient/donors pairs were analyzed using receiver operating characteristic (ROC) curve analyses to determine the accuracy of donor-specific median fluorescence intensity values (Σ MFI) from single antigen bead assays for predicting strong FC and CDC crossmatches. Diagnostic sensitivity and specificity of optimal Σ MFI values were highest for predicting strong T cell FCs. Σ MFI values showed good sensitivity for predicting positive direct and AHG-augmented CDC crossmatches (91% and 94%, respectively), but with lower specificity (67% each). Specificity and sensitivity for predicting positive B cell CDC crossmatches were 73% and 84%. Σ MFI values derived from single antigen bead assays can predict strong flow and positive CDC crossmatches, but with tradeoffs between sensitivity and specificity. The results support the use of solid phase assays for quantitative virtual crossmatching and as a replacement for cell-based crossmatching. [Copyright &y& Elsevier]

Regulatory T cell markers are increased in chronically infected individuals with the hepatitis C virus (HCV), but to date, the induction and maintenance of Tregs in HCV infection has not been clearly defined. In this paper, we demonstrate that naturally occurring viral variants suppress T cell responses to cognate NS3358-375 in an antigen-specific manner. Of four archetypal variants, S370P induced regulatory T cell markers in comparison to NS3358-375-stimulated CD4 T cells. Further, the addition of variantspecific CD4 T cells back into a polyclonal culture in a dose-dependent manner inhibited the T cell response. These results suggest thatHCV is able to induce antigen-specific regulatory T cells to suppress the antiviral T cell response in an antigen-specific manner, thus contributing to a niche within the host that could be conducive to HCV persistence. [ABSTRACT FROM AUTHOR]

The onset of type 1 diabetes can occur at any age, with as many as half of all cases diagnosed after age 15. Despite this wide distribution in age at diagnosis, most genetic studies focus on cases diagnosed in childhood or during early adulthood. To better understand the genetics of late-onset type 1 diabetes, we collected a Finnish case/control cohort with all cases diagnosed between ages 15 and 40. We genotyped 591 probands and 1,538 control subjects at regions well established as susceptibility loci in early onset type 1 diabetes. These loci were then tested for disease association and age-at-diagnosis effects. Using logistic regression, we found that single-nucleotide polymorphisms (SNPs) at the INS, PTPN22, and IFIH1 loci were associated with late-onset disease (OR (95%CI) = 0.57(0.47–0.69), p = 2.77 × 10−9; OR (95%CI) = 1.50 (1.27–1.78), p = 3.98 × 10−6; and OR (95%CI) = 0.81(0.71–0.93), p = 0.0028, respectively). In contrast, a disease association was not detected for two SNPs at the IL2RA locus (rs11594656 and rs41295061). Despite this, we did find an independent age-at-diagnosis effect for each IL2RA SNP using a multivariate Cox proportional hazards model ( p = 0.003, 0.002, respectively). Taken together, polymorphisms at the IL2RA locus were a major determinant of age at diagnosis in our cohort with an effect at par with the HLA-DQ2/DQ8 genotype as measured by hazard ratios. These findings suggest that the IL2RA locus controls both the susceptibility to disease and its time of occurrence. Thus, we believe the IL2/IL2R axis represents a potential therapeutic target for delaying the onset of disease. [ABSTRACT FROM AUTHOR]

Caspases play a critical role in the execution of metazoan apoptosis and are thus attractive therapeutic targets for apoptosis-associated diseases. Here we report that baculovirus P49, a homolog of pancaspase inhibitor P35, prevents apoptosis in invertebrates by inhibiting an initiator caspase that is P35 insensitive. Consequently P49 blocked proteolytic activation of effector caspases at a unique step upstream from that affected by P35 but downstream from inhibitor of apoptosis Op-lAP. Like P35, P49 was cleaved by and stably associated with its caspase target. Ectopically expressed P49 blocked apoptosis in cultured cells from a phylogenetically distinct organism, Drosophila melanogaster. Furthermore, P49 inhibited human caspase-9, demonstrating its capacity to affect a vertebrate initiator caspase. Thus, P49 is a substrate inhibitor with a novel in vivo specificity for a P35-insensitive initiator caspase that functions at an evolutionarily conserved step in the caspase cascade. These data indicate that activated initiator caspases provide another effective target for apoptotic intervention by substrate inhibitors. [ABSTRACT FROM AUTHOR]

Aim Sanger sequencing of HLA suffers from the inability to set phase in certain heterozygous combinations. NGS overcomes these problems, but the reliability is not well described. We tested the NGS TruSight® HLA Sequencing Panel (Illumina) for its ability to provide full length, genomic, accurate, unambiguous, phase-resolved HLA genotyping in a single assay on a panel of 145 specimens. Residual clinical, PT, and IHWG DNA samples from blood, buccal swabs, cell lines of varying quality, concentration, and age, were selected to cover every known antigen, as well as rare and novel alleles. Methods The 145 samples (2070 alleles) included 26 novel variants, 7 null alleles, and 7 PT samples. We used the Illumina NGS HLA genotyping system end-to-end, from Long-Range PCR and library preparation to sequencing on the MiSeq. FASTQ files were analyzed by Conexio Assign, providing phase-resolved genotyping results for HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1, -DPB1. We calculated concordance and unambiguous allele level identity in comparison to previously typed high resolution typed results (Sanger/SSP/SSO combined). Results 87.1% of all clusters (500 cycles) had a Q30 quality score (error rate of 0.1–0.01%). Concordance with original typing was 98.5%. Subdividing concordance, we found: 65.1% identical and unambiguous in reference and NGS; 20.8% unambiguous NGS, ambiguous reference; 9% unambiguous reference, ambiguous NGS; 3.7% ambiguous in both reference and NGS; and 1.5% discordant. Unambiguous reference/ambiguous NGS was the result of primer placement. Discordance (31 alleles) was due to novel alleles, reference errors, homopolymers and microsatellites, pseudogenes, and 3 instances of contamination. Benefits of NGS included discovery and resolution of novel alleles, identification and correction of IHWG reference and clinical mistypings. Analysis of 24 samples for 11 loci took ∼3–3.5 h. Conclusions TruSight® HLA Sequencing system is a reliable, accurate, comprehensive, ultra-high-resolution HLA typing method that can be easily implemented in the laboratory. For labs needing the highest resolution typing, it reduces the number of ancillary tests that must be performed to resolve ambiguities from ∼50% down to ∼14%. For registry labs, the concordance rate equals or exceeds 98.5%. F. Yamamoto: Grant/Research Support; Company/Organization; Illumina, Conexio. A. Lindell: Grant/Research Support; Company/Organization; Illumina, Conexio. B. Baas: Grant/Research Support; Company/Organization; Illumina, Conexio. A. Crawford: Grant/Research Support; Company/Organization; Illumina, Conexio. M. Won: Grant/Research Support; Company/Organization; Illumina, Conexio. N. Baird: Grant/Research Support; Company/Organization; Illumina, Conexio. M.W. Anderson: Grant/Research Support; Company/Organization; Illumina, Conexio. J. Nytes: Grant/Research Support; Company/Organization; Illumina, Conexio. J.J. Schiller: Grant/Research Support; Company/Organization; Illumina, Conexio. D. Goodridge: Grant/Research Support; Company/Organization; Illumina, Conexio. D.B. Tyan: Grant/Research Support; Company/Organization; Illumina, Conexio. [ABSTRACT FROM AUTHOR]

Aim: The FDA has issued new draft guidance on molecular testing around Research Use Only (RUO) and non-Quality System Regulation (QSR) products in clinical labs. Promega has introduced a new QSR-compliant nucleic acid extractor and kit specifically to address this issue. At BloodCenter of Wisconsin we have evaluated this new extractor, the Maxwell® CSC, in terms of its performance in HLA typing assays as well as its fit in the molecular HLA workflow. Methods: Patient samples (buccal swabs) were extracted using the Maxwell® CSC, and then evaluated for total yield and purity of DNA. They were compared to the existing extraction method and evaluated by sequence-based typing, rSSO and STR assays for overall success and results concordance. The data handling capabilities of the instrument were also evaluated, such as how the run reports affected technician time, downstream operations and integration with electronic records. Results: DNA concentrations from double-buccal swab isolations ranged from 13ng/μl to 247ng/μl, with a mean concentration of 76.31ng/μl. DNA purities ranged from OD260/280 ratios of 1.69 to 2.00, with a mean of 1.89. These results are comparable to those obtained on the Maxwell® MDx instrument in our laboratory. All samples tested in downstream HLA STR assay, KIR rSSO, HLA rSSO and sequence-based typing assays were successful and concordant with previously obtained results. Conclusions: The Maxwell® CSC is a QSR-compliant system which will allow molecular laboratories to adhere to the draft FDA guidance for molecular testing. It provides a solution for nucleic acid extraction which performs equivalently to Promega’s current extraction systems with regards to DNA yield, quality and utility in downstream assays. The Maxwell® CSC also helps reduce data entry tasks with exportable sample-specific results. This is useful for tracking samples throughout the workflow, reducing the need to re-enter sample identifiers, saving technicians time and reducing errors. [Copyright &y& Elsevier]