Publications by authors named "Jamie L Duke"

22 Publications

  • Page 1 of 1

HLA Class I Polymorphisms Influencing Both Peptide Binding and KIR Interactions Are Associated with Remission among Children with Atopic Dermatitis: A Longitudinal Study.

J Immunol 2021 05 16;206(9):2038-2044. Epub 2021 Apr 16.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA;

Atopic dermatitis (AD) is a disease of immune dysregulation and skin barrier dysfunction with a relapsing, remitting course and has been associated with several different genetic risk variants. HLA represent a highly variable set of genes that code for cell surface protein molecules involved in the Ag-specific immune response, including the regulation or functioning of T cells, NK cells, and APCs. The purpose of this study was to evaluate associations between HLA class I polymorphisms and the progression of AD over time. We evaluated the associations of AD symptoms and HLA class I polymorphisms based on high-resolution two-field typing in a longitudinal cohort of children with AD (up to 10 y of follow-up). Seven hundred and ninety-two children were evaluated every 6 mo, resulting in 12,752 AD evaluations. Using generalized estimating equations and corrected values, B*44:02 was found to be associated with AD remission (1.83 [1.35, 2.47]; = 0.0015). The HLA-B residues at position 116 (d-aspartate) and 80 (T-threonine) were associated with remission (1.42 [1.13, 1.76], = 0.003; corrected = 0.028) and (1.45 [1.17, 1.80], = 0.0008; corrected = 0.0024), respectively. B80T is a killer-cell Ig-like receptor (KIR) site. Our findings reveal that two axes of immune response (T cell and NK cell) may influence disease progression. Identifying binding pocket changes in addition to other factors (e.g., allergens) that increase the risk or severity of AD can improve our understanding of the immunologic mechanisms associated with AD and may lead to personalized therapies for improving patient care.
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http://dx.doi.org/10.4049/jimmunol.2001252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062288PMC
May 2021

Spatial constrains and information content of sub-genomic regions of the human genome.

iScience 2021 Feb 10;24(2):102048. Epub 2021 Jan 10.

Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Complexity metrics and machine learning (ML) models have been utilized to analyze the lengths of segmental genomic entities of DNA sequences (exonic, intronic, intergenic, repeat, unique) with the purpose to ask questions regarding the segmental organization of the human genome within the size distribution of these sequences. For this we developed an integrated methodology that is based upon the reconstructed phase space theorem, the non-extensive statistical theory of Tsallis, ML techniques, and a technical index, integrating the generated information, which we introduce and named complexity factor (COFA). Our analysis revealed that the size distribution of the genomic regions within chromosomes are not random but follow patterns with characteristic features that have been seen through its complexity character, and it is part of the dynamics of the whole genome. Finally, this picture of dynamics in DNA is recognized using ML tools for clustering, classification, and prediction with high accuracy.
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http://dx.doi.org/10.1016/j.isci.2021.102048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7843455PMC
February 2021

Genomic characterization of MICA gene using multiple next generation sequencing platforms: A validation study.

HLA 2020 10 21;96(4):430-444. Epub 2020 Aug 21.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

We have developed a protocol regarding the genomic characterization of the MICA gene by next generation sequencing (NGS). The amplicon includes the full length of the gene and is about 13 kb. A total of 156 samples were included in the study. Ninety-seven of these samples were previously characterized at MICA by legacy methods (Sanger or sequence specific oligonucleotide) and were used to evaluate the accuracy, precision, specificity, and sensitivity of the assay. An additional 59 DNA samples of unknown ethnicity volunteers from the United States were only genotyped by NGS. Samples were chosen to contain a diverse set of alleles. Our NGS approach included a first round of sequencing on the Illumina MiSeq platform and a second round of sequencing on the MinION platform by Oxford Nanopore Technology (ONT), on selected samples for the purpose of either characterizing new alleles or setting phase among multiple polymorphisms to resolve ambiguities or generate complete sequence for alleles that were only partially reported in the IMGT/HLA database. Complete consensus sequences were generated for every allele sequenced with ONT, extending from the 5' untranslated region (UTR) to the 3' UTR of the MICA gene. Thirty-two MICA sequences were submitted to the IMGT/HLA database including either new alleles or filling up the gaps (exonic, intronic and/or UTRs) of already reported alleles. Some of the challenges associated with the characterization of these samples are discussed.
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http://dx.doi.org/10.1111/tan.13998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589345PMC
October 2020

Utilizing nanopore sequencing technology for the rapid and comprehensive characterization of eleven HLA loci; addressing the need for deceased donor expedited HLA typing.

Hum Immunol 2020 Aug 25;81(8):413-422. Epub 2020 Jun 25.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

The comprehensive characterization of human leukocyte antigen (HLA) genomic sequences remains a challenging problem. Despite the significant advantages of next-generation sequencing (NGS) in the field of Immunogenetics, there has yet to be a single solution for unambiguous, accurate, simple, cost-effective, and timely genotyping necessary for all clinical applications. This report demonstrates the benefits of nanopore sequencing introduced by Oxford Nanopore Technologies (ONT) for HLA genotyping. Samples (n = 120) previously characterized at high-resolution three-field (HR-3F) for 11 loci were assessed using ONT sequencing paired to a single-plex PCR protocol (Holotype) and to two multiplex protocols OmniType (Omixon) and NGSgo®-MX6-1 (GenDx). The results demonstrate the potential of nanopore sequencing for delivering accurate HR-3F typing with a simple, rapid, and cost-effective protocol. The protocol is applicable to time-sensitive applications, such as deceased donor typings, enabling better assessments of compatibility and epitope analysis. The technology also allows significantly shorter turnaround time for multiple samples at a lower cost. Overall, the nanopore technology appears to offer a significant advancement over current next-generation sequencing platforms as a single solution for all HLA genotyping needs.
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http://dx.doi.org/10.1016/j.humimm.2020.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870017PMC
August 2020

Complex Linkage Disequilibrium Effects in HLA-DPB1 Expression and Molecular Mismatch Analyses of Transplantation Outcomes.

Transplantation 2021 03;105(3):637-647

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA.

Background: HLA molecular mismatch (MM) is a risk factor for de novo donor-specific antibody (dnDSA) development in solid organ transplantation. HLA expression differences have also been associated with adverse outcomes in hematopoietic cell transplantation. We sought to study both MM and expression in assessing dnDSA risk.

Methods: One hundred three HLA-DP-mismatched solid organ transplantation pairs were retrospectively analyzed. MM was computed using amino acids (aa), eplets, and, supplementarily, Grantham/Epstein scores. DPB1 alleles were classified as rs9277534-A (low-expression) or rs9277534-G (high-expression) linked. To determine the associations between risk factors and dnDSA, logistic regression, linkage disequilibrium (LD), and population-based analyses were performed.

Results: A high-risk AA:GX (recipient:donor) expression combination (X = A or G) demonstrated strong association with HLA-DP dnDSA (P = 0.001). MM was also associated with HLA-DP dnDSA when evaluated by itself (eplet P = 0.007, aa P = 0.003, Grantham P = 0.005, Epstein P = 0.004). When attempting to determine the relative individual effects of the risk factors in multivariable analysis, only AA:GX expression status retained a strong association (relative risk = 18.6, P = 0.007 with eplet; relative risk = 15.8, P = 0.02 with aa), while MM was no longer significant (eplet P = 0.56, aa P = 0.51). Importantly, these risk factors are correlated, due to LD between the expression-tagging single-nucleotide polymorphism and polymorphisms along HLA-DPB1.

Conclusions: The MM and expression risk factors each appear to be strong predictors of HLA-DP dnDSA and to possess clinical utility; however, these two risk factors are closely correlated. These metrics may represent distinct ways of characterizing a common overlapping dnDSA risk profile, but they are not independent. Further, we demonstrate the importance and detailed implications of LD effects in dnDSA risk assessment and possibly transplantation overall.
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http://dx.doi.org/10.1097/TP.0000000000003272DOI Listing
March 2021

Resolving MiSeq-Generated Ambiguities in HLA-DPB1 Typing by Using the Oxford Nanopore Technology.

J Mol Diagn 2019 09 4;21(5):852-861. Epub 2019 Jun 4.

Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

The technical limitations of current next-generation sequencing technologies, combined with an ever-increasing number of human leukocyte antigen (HLA) alleles, form the basis for the additional ambiguities encountered at an increasing rate in clinical practice. HLA-DPB1 characterization, particularly, generates a significant percentage of ambiguities (25.5%), posing a challenge for accurate and unambiguous HLA-DPB1 genotyping. Phasing of exonic heterozygous positions between exon 2 and all other downstream exons has been the major cause of ambiguities. In this study, the Oxford Nanopore MinION, a third-generation sequencing technology, was used to resolve the phasing. The accurate MiSeq sequencing data, combined with the long reads obtained from the MinION platform, allow for the resolution of the tested ambiguities.
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http://dx.doi.org/10.1016/j.jmoldx.2019.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6734860PMC
September 2019

Development of hemolytic paroxysmal nocturnal hemoglobinuria without graft loss following hematopoietic stem cell transplantation for acquired aplastic anemia.

Pediatr Transplant 2019 06 22;23(4):e13393. Epub 2019 Mar 22.

Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

PNH is the most common clonal hematopoietic disorder arising in patients with aAA. PNH is caused by mutations in PIGA, a gene that encodes the catalytic subunit of an enzyme involved in the biosynthesis of GPI anchors, transmembrane glycolipids required for cell surface expression of many proteins. PNH clones likely arise as immune escape mechanisms in aAA by preventing CD1D-restricted T-cell recognition of GPI anchors and GPI-linked autoantigens. Though many patients with aAA treated with IST will develop subclinical PNH clones, only a subset will develop PNH disease, characterized by increased thrombosis, intravascular hemolysis, and potential for severe organ dysfunction. In contrast to IST, allogeneic HSCT for patients with aAA is thought to cure bone marrow aplasia and prevent hematopoietic clonal evolution to PNH. Herein, we present a phenomenon of host-derived PNH disease arising in a patient with aAA many years following MSD-BMT, highlighting the importance of monitoring for this clonal disease in aAA patients with stable mixed donor/recipient chimerism after HSCT. We also provide a literature review for similar occurrences of PNH arising after HSCT.
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http://dx.doi.org/10.1111/petr.13393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548609PMC
June 2019

Next-generation HLA typing of 382 International Histocompatibility Working Group reference B-lymphoblastoid cell lines: Report from the 17th International HLA and Immunogenetics Workshop.

Hum Immunol 2019 Jul 4;80(7):449-460. Epub 2019 Mar 4.

Department of Pathology, University of California San Diego, La Jolla, CA, USA.

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.
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http://dx.doi.org/10.1016/j.humimm.2019.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599558PMC
July 2019

Assessing the utilization of high-resolution 2-field HLA typing in solid organ transplantation.

Am J Transplant 2019 07 22;19(7):1955-1963. Epub 2019 Feb 22.

Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

HLA typing in solid organ transplantation (SOT) is necessary for determining HLA-matching status between donor-recipient pairs and assessing patients' anti-HLA antibody profiles. Histocompatibility has traditionally been evaluated based on serologically defined HLA antigens. The evolution of HLA typing and antibody identification technologies, however, has revealed many limitations with using serologic equivalents for assessing compatibility in SOT. The significant improvements to HLA typing introduced by next-generation sequencing (NGS) require an assessment of the impact of this technology on SOT. We have assessed the role of high-resolution 2-field HLA typing (HR-2F) in SOT by retrospectively evaluating NGS-typed pre- and post-SOT cases. HR-2F typing was highly instructive or necessary in 41% (156/385) of the cases. Several pre- and posttransplant scenarios were identified as being better served by HR-2F typing. Five different categories are presented with specific case examples. The experience of another center (Temple University Hospital) is also included, whereby 21% of the cases required HR-2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti-HLA antibody issues.
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http://dx.doi.org/10.1111/ajt.15258DOI Listing
July 2019

Characterization of 108 Genomic DNA Reference Materials for 11 Human Leukocyte Antigen Loci: A GeT-RM Collaborative Project.

J Mol Diagn 2018 09 26;20(5):703-715. Epub 2018 Jun 26.

Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

The highly polymorphic human leukocyte antigen (HLA) genes, located in the human major histocompatibility complex, encode the class I and II antigen-presenting molecules, which are centrally involved in the immune response. HLA typing is used for several clinical applications, such as transplantation, pharmacogenetics, and diagnosis of autoimmune disease. HLA typing is highly complex because of the homology of HLA genes and pseudogenes and the extensive polymorphism in the population. The Centers for Disease Control and Prevention established the Genetic Testing Reference Materials Coordination Program (GeT-RM) in partnership with the genetics community to improve the availability of genomic DNA reference materials necessary for quality assurance of genetic laboratory testing. The GeT-RM together with three clinical laboratories and the Coriell Cell Repositories have characterized genomic DNA obtained from a panel of 108 cell lines for all HLA classic polymorphic loci: HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, and DPB1. The goal was to develop a publicly available and renewable source of well-characterized genomic DNA reference materials to support molecular HLA typing assay development, validation, and verification, quality control, and proficiency testing. These genomic DNA samples are publicly available from the National Institutes of General Medical Science Repository at the Coriell Cell Repositories.
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http://dx.doi.org/10.1016/j.jmoldx.2018.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939753PMC
September 2018

Somatic HLA Mutations Expose the Role of Class I-Mediated Autoimmunity in Aplastic Anemia and its Clonal Complications.

Blood Adv 2017 Oct;1(22):1900-1910

Comprehensive Bone Marrow Failure Center, Department of Pediatrics, Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104.

Acquired aplastic anemia (aAA) is an acquired deficiency of early hematopoietic cells, characterized by inadequate blood production, and a predisposition to myelodysplastic syndrome (MDS) and leukemia. Although its exact pathogenesis is unknown, aAA is thought to be driven by Human Leukocyte Antigen (HLA)-restricted T cell immunity, with earlier studies favoring HLA class II-mediated pathways. Using whole exome sequencing (WES), we recently identified two aAA patients with somatic mutations in HLA class I genes. We hypothesized that HLA class I mutations are pathognomonic for autoimmunity in aAA, but were previously underappreciated because the Major Histocompatibility Complex (MHC) region is notoriously difficult to analyze by WES. Using a combination of targeted deep sequencing of HLA class I genes and single nucleotide polymorphism array (SNP-A) genotyping we screened 66 aAA patients for somatic HLA class I loss. We found somatic HLA loss in eleven patients (17%), with thirteen loss-of-function mutations in *33:03, *68:01, *14:02 and *40:02 alleles. Three patients had more than one mutation targeting the same HLA allele. Interestingly, *14:02 and *40:02 were significantly overrepresented in aAA patients, compared to ethnicity-matched controls. Patients who inherited the targeted HLA alleles, regardless of HLA mutation status, had a more severe disease course with more frequent clonal complications as assessed by WES, SNP-A, and metaphase cytogenetics, and more frequent secondary MDS. The finding of recurrent HLA class I mutations provides compelling evidence for a predominant HLA class I-driven autoimmunity in aAA, and establishes a novel link between aAA patients' immunogenetics and clonal evolution.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621748PMC
http://dx.doi.org/10.1182/bloodadvances.2017010918DOI Listing
October 2017

Assessing a single targeted next generation sequencing for human leukocyte antigen typing protocol for interoperability, as performed by users with variable experience.

Hum Immunol 2017 Oct 18;78(10):642-648. Epub 2017 Jul 18.

Cleveland Clinic, United States; Baylor University Medical Center, United States.

Background: A simplified protocol for HLA-typing -by NGS, developed for use with the Illumina MiSeq, was performed by technologists with varying NGS experience to assess accuracy and reproducibility.

Methods: Technologists from six laboratories typed the same 16 samples at HLA-A, B, C, DRB1, and DQB1. The protocol includes long range PCR, library preparation, and paired-end 250bp sequencing. Two indexing strategies were employed: locus-specific indexing whereby each locus was tagged uniquely and sample-specific indexing whereby all 5 loci for a sample were pooled prior to library preparation. Sequence analysis was performed with two software packages, Target HLA (Omixon) and NGSengine (GenDx).

Results: The average number of sequence reads per library was 387,813; however, analysis was limited to 40,000 reads for locus-indexed libraries and 200,000 reads for sample-indexed libraries resulting in an average depth of coverage of 1444 reads per locus. Sufficient reads for genotype analysis were obtained for 98.4% of libraries. Genotype accuracy was >97% in pooled amplicons and >99% in individual amplicons by both software analysis. Inter-laboratory reproducibility was 99.7% and only cause of discordance was cross-contamination of a single amplicon.

Conclusions: This NGS HLA-typing protocol is simple, reproducible, scalable, highly accurate and amenable to clinical testing.
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http://dx.doi.org/10.1016/j.humimm.2017.07.012DOI Listing
October 2017

Targeted Next-Generation Sequencing for Human Leukocyte Antigen Typing in a Clinical Laboratory: Metrics of Relevance and Considerations for Its Successful Implementation.

Arch Pathol Lab Med 2017 Jun 24;141(6):806-812. Epub 2017 Feb 24.

From the Division of Transfusion Medicine, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota (Dr Gandhi); the Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania (Ms Ferriola and Drs Huang, Duke, and Monos); and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia (Dr Monos).

Context: - Numerous feasibility studies to type human leukocyte antigens (HLAs) by next-generation sequencing (NGS) have led to the development of vendor-supported kits for HLA typing by NGS. Some clinical laboratories have introduced HLA-NGS, and many are investigating the introduction. Standards from accrediting agencies form the regulatory framework for introducing this test into clinical laboratories.

Objectives: - To provide an assessment of metrics and considerations relevant to the successful implementation of clinical HLA-NGS typing, and to provide as a reference a validated HLA-NGS protocol used clinically since December 2013 at the Children's Hospital of Philadelphia (Philadelphia, Pennsylvania).

Data Sources: - The HLA-NGS has been performed on 2532 samples. The initial 1046 and all homozygous samples were also typed by an alternate method. The HLA-NGS demonstrated 99.7% concordance with the alternate method. Ambiguous results were most common at the DPB1 locus because of a lack of phasing between exons 2 and 3 or the unsequenced exon 1 (533 of 2954 alleles; 18.04%) and the DRB1 locus because of not sequencing exon 1 (75 of 3972 alleles; 1.89%). No ambiguities were detected among the other loci. Except for 2 false homozygous samples, all homozygous samples (1891) demonstrated concordance with the alternate method. The article is organized to address the critical elements in the preanalytic, analytic, and postanalytic phases of introducing this assay into the clinical laboratory.

Conclusions: - The results demonstrate that HLA typing by NGS is a highly accurate, reproducible, efficient method that provides more-complete sequencing information for the length of the HLA gene and can be the single methodology for HLA typing in clinical immunogenetics laboratories.
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http://dx.doi.org/10.5858/arpa.2016-0537-RADOI Listing
June 2017

Generation of Full-Length Class I Human Leukocyte Antigen Gene Consensus Sequences for Novel Allele Characterization.

Clin Chem 2016 Dec 27;62(12):1630-1638. Epub 2016 Sep 27.

Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA;

Background: Routine, high-resolution human leukocyte antigen (HLA) genotyping by next generation sequencing within clinical immunogenetics laboratories can now provide the full-length gene sequence characterization of fully phased HLA alleles. This powerful technique provides insights into HLA variation beyond the traditionally characterized antigen recognition domain, providing sequence annotation across the entire gene including untranslated and intronic regions and may be used to characterize novel alleles from massively parallel sequencing runs.

Methods: We evaluated the utility of the Omixon Holotype HLA assay to generate credible, fully phased full-length gene consensus sequences for 50 individuals at major histocompatibility complex, class I, A (HLA-A), HLA-B, and HLA-C loci (300 genotyped alleles in total) to identify and characterize novel class I HLA alleles using our downstream analytical pipeline.

Results: Our analysis revealed that 7.7% (23/300) of genotyped class I HLA alleles contain novel polymorphisms. Interestingly, all of the novel alleles identified by our analysis were found to harbor sequence variations within intronic regions of the respective locus. In total our analysis identified 17 unique novel class I HLA alleles from 23 of the 300 genotyped alleles and generated full-length gene sequence annotations for 9 previously incompletely annotated HLA class I allele sequences derived from 14 of the 300 genotyped alleles.

Conclusions: The demonstrated utility of the Omixon Holotype HLA assay in combination with our downstream analytical framework to generate fully phased, full-length gene consensus sequences for the identification and characterization of novel HLA alleles, facilitates the study of HLA polymorphism beyond the antigen recognition domain in human health and disease.
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http://dx.doi.org/10.1373/clinchem.2016.260661DOI Listing
December 2016

Association of HLA-DRB1 genetic variants with the persistence of atopic dermatitis.

Hum Immunol 2015 Aug 22;76(8):571-7. Epub 2015 Aug 22.

Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, United States.

Atopic dermatitis (AD) is a waxing and waning illness of childhood that is likely caused by interactions between an altered skin barrier and immune dysregulation. The goal of our study was to evaluate the association of DRB1 genetic variants and the persistence of AD using whole exome sequencing and high resolution typing. DRB1 was interrogated based on previous reports that utilized high throughput techniques. We evaluated an ongoing nation-wide long-term cohort of children with AD in which patients are asked every 6months about their medication use and their AD symptoms. In total, 87 African-American and 50 European-American children were evaluated. Genetic association analysis was performed using a software tool focusing on amino acid variable positions shared by HLA-DRB1 alleles covering the antigen presenting domain. Amino acid variations at position 9 (pocket 9), position 26, and position 78 (pocket 4) were marginally associated with the prevalence of AD. However, the odds ratio was 0.30 (0.14, 0.68; p=0.003) for residue 78, 0.27 (0.10, 0.69; p=0.006) for residue 26 and not significant for residue 9 with respect to the persistence of AD. In conclusion, amino acid variations at peptide-binding pockets of HLA-DRB1 were associated with the persistence of AD in African-American children.
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http://dx.doi.org/10.1016/j.humimm.2015.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4593755PMC
August 2015

Overcoming NS1-mediated immune antagonism involves both interferon-dependent and independent mechanisms.

J Interferon Cytokine Res 2013 Nov 17;33(11):700-8. Epub 2013 Jun 17.

1 Department of Pathology, Yale School of Medicine , New Haven, Connecticut.

To ensure survival, our immune system must overcome the action of pathogen-encoded immune antagonists, such as influenza A nonstructural protein-1 (NS1). NS1 subverts the host interferon (IFN) response at multiple levels and blocks the induction of IFN-β, a critical antiviral cytokine. This immune antagonism can be overcome in some cases. It has been shown that IFN-β is upregulated by 48 h in the lungs of wild-type C57BL/6 mice infected with influenza A. In contrast, it is shown here that IFNB1 continues to be repressed in IFNAR1(-/-) IL28Rα(-/-) mice, which are deficient in Type-I and III IFN signaling, implying induction of IFNB1 depends on effective IFN signaling. Despite the complete lack of IFN signaling in this system, some IFN stimulated genes (ISGs) were induced following infection with a Flu strain lacking NS1. While the expression of these viral stress-inducible genes (VSIGs) was initially repressed following infection with wild-type Flu, many of these genes became upregulated by 48 h postinfection. These results demonstrate the existence of IFN-independent mechanisms that can overcome NS1-mediated immune antagonism of VSIGs.
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http://dx.doi.org/10.1089/jir.2012.0113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814816PMC
November 2013

Multiple transcription factor binding sites predict AID targeting in non-Ig genes.

J Immunol 2013 Apr 20;190(8):3878-88. Epub 2013 Mar 20.

Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT 06511, USA.

Aberrant targeting of the enzyme activation-induced cytidine deaminase (AID) results in the accumulation of somatic mutations in ≈ 25% of expressed genes in germinal center B cells. Observations in Ung(-/-) Msh2(-/-) mice suggest that many other genes efficiently repair AID-induced lesions, so that up to 45% of genes may actually be targeted by AID. It is important to understand the mechanisms that recruit AID to certain genes, because this mistargeting represents an important risk for genome instability. We hypothesize that several mechanisms combine to target AID to each locus. To resolve which mechanisms affect AID targeting, we analyzed 7.3 Mb of sequence data, along with the regulatory context, from 83 genes in Ung(-/-) Msh2(-/-) mice to identify common properties of AID targets. This analysis identifies three transcription factor binding sites (E-box motifs, along with YY1 and C/EBP-β binding sites) that may work together to recruit AID. Based on previous knowledge and these newly discovered features, a classification tree model was built to predict genome-wide AID targeting. Using this predictive model, we were able to identify a set of 101 high-interest genes that are likely targets of AID.
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http://dx.doi.org/10.4049/jimmunol.1202547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3689293PMC
April 2013

Identification of core DNA elements that target somatic hypermutation.

J Immunol 2012 Dec 19;189(11):5314-26. Epub 2012 Oct 19.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.

Somatic hypermutation (SHM) diversifies the V region of Ig genes and underlies the process of affinity maturation, in which B lymphocytes producing high-affinity Abs are generated and selected. SHM is triggered in activated B cells by deamination of deoxycytosine residues mediated by activation-induced deaminase (AID). Whereas mistargeting of SHM and AID results in mutations and DNA damage in many non-Ig genes, they act preferentially at Ig loci. The mechanisms responsible for preferential targeting of SHM and AID activity to Ig loci are poorly understood. Using an assay involving an SHM reporter cassette inserted into the Ig L chain locus (IgL) of chicken DT40 B cells, we have identified a 1.9-kb DIVAC (diversification activator) element derived from chicken IgL that supports high levels of AID-dependent mutation activity. Systematic deletion analysis reveals that targeting activity is spread throughout much of the sequence and identifies two core regions that are particularly critical for function: a 200-bp region within the IgL enhancer, and a 350-bp 3' element. Chromatin immunoprecipitation experiments demonstrate that whereas DIVAC does not alter levels of several epigenetic marks in the mutation cassette, it does increase levels of serine-5 phosphorylated RNA polymerase II in the mutation target region, consistent with an effect on transcriptional elongation/pausing. We propose that multiple, dispersed DNA elements collaborate to recruit and activate the mutational machinery at Ig gene variable regions during SHM.
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http://dx.doi.org/10.4049/jimmunol.1202082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664039PMC
December 2012

Gene expression analysis of forskolin treated basilar papillae identifies microRNA181a as a mediator of proliferation.

PLoS One 2010 Jul 9;5(7):e11502. Epub 2010 Jul 9.

Medical Scientist Training Program, Yale School of Medicine, New Haven, Connecticut, United States of America.

Background: Auditory hair cells spontaneously regenerate following injury in birds but not mammals. A better understanding of the molecular events underlying hair cell regeneration in birds may allow for identification and eventually manipulation of relevant pathways in mammals to stimulate regeneration and restore hearing in deaf patients.

Methodology/principal Findings: Gene expression was profiled in forskolin treated (i.e., proliferating) and quiescent control auditory epithelia of post-hatch chicks using an Affymetrix whole-genome chicken array after 24 (n = 6), 48 (n = 6), and 72 (n = 12) hours in culture. In the forskolin-treated epithelia there was significant (p<0.05; >two-fold change) upregulation of many genes thought to be relevant to cell cycle control and inner ear development. Gene set enrichment analysis was performed on the data and identified myriad microRNAs that are likely to be upregulated in the regenerating tissue, including microRNA181a (miR181a), which is known to mediate proliferation in other systems. Functional experiments showed that miR181a overexpression is sufficient to stimulate proliferation within the basilar papilla, as assayed by BrdU incorporation. Further, some of the newly produced cells express the early hair cell marker myosin VI, suggesting that miR181a transfection can result in the production of new hair cells.

Conclusions/significance: These studies have identified a single microRNA, miR181a, that can cause proliferation in the chicken auditory epithelium with production of new hair cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011502PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901389PMC
July 2010

Coregulation mapping based on individual phenotypic variation in response to virus infection.

Immunome Res 2010 Mar 18;6. Epub 2010 Mar 18.

Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA.

Background: Gene coregulation across a population is an important aspect of the considerable variability of the human immune response to virus infection. Methodology to investigate it must rely on a number of ingredients ranging from gene clustering to transcription factor enrichment analysis.

Results: We have developed a methodology to investigate the gene to gene correlations for the expression of 34 genes linked to the immune response of Newcastle Disease Virus (NDV) infected conventional dendritic cells (DCs) from 145 human donors. The levels of gene expression showed a large variation across individuals. We generated a map of gene co-expression using pairwise correlation and multidimensional scaling (MDS). The analysis of these data showed that among the 13 genes left after filtering for statistically significant variations, two clusters are formed. We investigated to what extent the observed correlation patterns can be explained by the sharing of transcription factors (TFs) controlling these genes. Our analysis showed that there was a significant positive correlation between MDS distances and TF sharing across all pairs of genes. We applied enrichment analysis to the TFs having binding sites in the promoter regions of those genes. This analysis, after Gene Ontology filtering, indicated the existence of two clusters of genes (CCL5, IFNA1, IFNA2, IFNB1) and (IKBKE, IL6, IRF7, MX1) that were transcriptionally co-regulated. In order to facilitate the use of our methodology by other researchers, we have also developed an interactive coregulation explorer web-based tool called CorEx. It permits the study of MDS and hierarchical clustering of data combined with TF enrichment analysis. We also offer web services that provide programmatic access to MDS, hierarchical clustering and TF enrichment analysis.

Conclusions: MDS mapping based on correlation in conjunction with TF enrichment analysis represents a useful computational method to generate predictions underlying gene coregulation across a population.
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http://dx.doi.org/10.1186/1745-7580-6-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161383PMC
March 2010

Antiviral response dictated by choreographed cascade of transcription factors.

J Immunol 2010 Mar 17;184(6):2908-17. Epub 2010 Feb 17.

Center for Translational Systems Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.

The dendritic cell (DC) is a master regulator of immune responses. Pathogenic viruses subvert normal immune function in DCs through the expression of immune antagonists. Understanding how these antagonists interact with the host immune system requires knowledge of the underlying genetic regulatory network that operates during an uninhibited antiviral response. To isolate and identify this network, we studied DCs infected with Newcastle disease virus, which is able to stimulate innate immunity and DC maturation through activation of RIG-I signaling, but lacks the ability to evade the human IFN response. To analyze this experimental model, we developed a new approach integrating genome-wide expression kinetics and time-dependent promoter analysis. We found that the genetic program underlying the antiviral cell-state transition during the first 18 h postinfection could be explained by a single convergent regulatory network. Gene expression changes were driven by a stepwise multifactor cascading control mechanism, where the specific transcription factors controlling expression changed over time. Within this network, most individual genes were regulated by multiple factors, indicating robustness against virus-encoded immune evasion genes. In addition to effectively recapitulating current biological knowledge, we predicted, and validated experimentally, antiviral roles for several novel transcription factors. More generally, our results show how a genetic program can be temporally controlled through a single regulatory network to achieve the large-scale genetic reprogramming characteristic of cell-state transitions.
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http://dx.doi.org/10.4049/jimmunol.0903453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2856074PMC
March 2010

Two levels of protection for the B cell genome during somatic hypermutation.

Nature 2008 Feb;451(7180):841-5

Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

Somatic hypermutation introduces point mutations into immunoglobulin genes in germinal centre B cells during an immune response. The reaction is initiated by cytosine deamination by the activation-induced deaminase (AID) and completed by error-prone processing of the resulting uracils by mismatch and base excision repair factors. Somatic hypermutation represents a threat to genome integrity and it is not known how the B cell genome is protected from the mutagenic effects of somatic hypermutation nor how often these protective mechanisms fail. Here we show, by extensive sequencing of murine B cell genes, that the genome is protected by two distinct mechanisms: selective targeting of AID and gene-specific, high-fidelity repair of AID-generated uracils. Numerous genes linked to B cell tumorigenesis, including Myc, Pim1, Pax5, Ocab (also called Pou2af1), H2afx, Rhoh and Ebf1, are deaminated by AID but escape acquisition of most mutations through the combined action of mismatch and base excision repair. However, approximately 25% of expressed genes analysed were not fully protected by either mechanism and accumulated mutations in germinal centre B cells. Our results demonstrate that AID acts broadly on the genome, with the ultimate distribution of mutations determined by a balance between high-fidelity and error-prone DNA repair.
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http://dx.doi.org/10.1038/nature06547DOI Listing
February 2008
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