42 results on '"Heusel JW"'
Search Results
2. High-depth next-generation sequencing panel testing in the evaluation of arteriovenous malformations.
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Hernandez PV, King KA, Evenson MJ, Corliss MM, Schroeder MC, Heusel JW, Neidich JA, and Cao Y
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- Humans, Female, Male, Mutation, Germ-Line Mutation, Chromosome Aberrations, High-Throughput Nucleotide Sequencing methods, p120 GTPase Activating Protein genetics, Arteriovenous Malformations diagnosis, Arteriovenous Malformations genetics
- Abstract
Arteriovenous malformations (AVMs) are vascular lesions in which an overgrowth of blood vessels of varying sizes develops with one or more direct connections between the arterial and venous circulation. We performed a retrospective review of a cohort of 54 patients with AVMs referred to our clinical genomic laboratory for high-depth next-generation sequencing (NGS) panel of Disorders of Somatic Mosaicism (DoSM). Thirty-seven of 54 patients were female (68.5%). Among the 54 cases, 37 (68.5%) cases had pathogenic and/or likely pathogenic (P/LP) variants identified, two cases (3.7%) had variants of uncertain clinical significance, and the remaining 15 cases (27.8%) had negative results. MAP2K1 variants were found in 12 cases, followed by eight cases with KRAS variants and seven with TEK variants, and the remainder being identified in several other genes on the panel. Among the 37 positive cases, 32 cases had somatic alterations only; the remaining five cases had at least one germline P/LP variant, including four cases with PTEN and one with RASA1. Of note, two cases had the unexpected co-existence of two P/LP variants. In summary, this study illustrated the molecular diagnostic yield (68.5%) of this cohort of patients with a clinical indication of AVMs by our high-depth DoSM NGS panel., (© 2023 Wiley Periodicals LLC.)
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- 2023
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3. A comparative analysis of RAS variants in patients with disorders of somatic mosaicism.
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Claire Hou YC, Evenson MJ, Corliss MM, Mahapatra L, Aldawood A, Carpentieri DF, Chamlin SL, Kulungowski AM, Madan-Khetarpal S, Sebastian J, Pet MA, Coughlin CC, Willing MC, Pearson GD, Setty BA, El-Haffaf Z, Cottrell CE, Parikh BA, Krysiak K, Schroeder MC, Heusel JW, Neidich JA, and Cao Y
- Subjects
- Humans, Proto-Oncogene Proteins p21(ras) genetics, Mutation, Alleles, Mosaicism, Vascular Malformations genetics
- Abstract
Purpose: RAS genes (HRAS, KRAS, and NRAS) are commonly found to be mutated in cancers, and activating RAS variants are also found in disorders of somatic mosaicism (DoSM). A survey of the mutational spectrum of RAS variants in DoSM has not been performed., Methods: A total of 938 individuals with suspected DoSM underwent high-sensitivity clinical next-generation sequencing-based testing. We investigated the mutational spectrum and genotype-phenotype associations of mosaic RAS variants., Results: In this article, we present a series of individuals with DoSM with RAS variants. Classic hotspots, including Gly12, Gly13, and Gln61 constituted the majority of RAS variants observed in DoSM. Furthermore, we present 12 individuals with HRAS and KRAS in-frame duplication/insertion (dup/ins) variants in the switch II domain. Among the 18.3% individuals with RAS in-frame dup/ins variants, clinical findings were mainly associated with vascular malformations. Hotspots were associated with a broad phenotypic spectrum, including vascular tumors, vascular malformations, nevoid proliferations, segmental overgrowth, digital anomalies, and combinations of these. The median age at testing was higher and the variant allelic fraction was lower in individuals with in-frame dup/ins variants than those in individuals with mosaic RAS hotspots., Conclusion: Our work provides insight into the allelic and clinical heterogeneity of mosaic RAS variants in nonmalignant conditions., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)
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- 2023
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4. Multivariate analysis of associations between clinical sequencing and outcome in glioblastoma.
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Yang PH, Tao Y, Luo J, Paturu M, Lu HC, Ramkissoon S, Heusel JW, Leuthardt EC, Chicoine MR, Dowling JL, Dunn GP, Duncavage E, Dahiya S, Chattherjee AR, and Kim AH
- Abstract
Background: Many factors impact survival in patients with glioblastoma, including age, Karnofsky Performance Status, postoperative chemoradiation, IDH1/2 mutation status, MGMT promoter methylation status, and extent of resection. High-throughput next-generation sequencing is a widely available diagnostic tool, but the independent impact of tumors harboring specific mutant genes on survival and the efficacy of extent of resection are not clear., Methods: We utilized a widely available diagnostic platform (FoundationOne CDx) to perform high-throughput next-generation sequencing on 185 patients with newly diagnosed glioblastoma in our tertiary care center. We performed multivariate analysis to control for clinical parameters with known impact on survival to elucidate the independent prognostic value of prevalent mutant genes and the independent impact of gross total resection., Results: When controlling for factors with known prognostic significance including IDH1/2 mutation and after multiple comparisons analysis, CDKN2B and EGFR mutations were associated with reduced overall survival while PTEN mutation was associated with improved overall survival. Gross total resection, compared to other extent of resection, was associated with improved overall survival in patients with tumors harboring mutations in CDKN2A , CDKN2B , EGFR , PTEN , TERT promoter, and TP53 . All patients possessed at least one of these 6 mutant genes., Conclusions: This study verifies the independent prognostic value of several mutant genes in glioblastoma. Six commonly found mutant genes were associated with improved survival when gross total resection was achieved. Thus, even when accounting for known predictors of survival and multiple mutant gene comparisons, extent of resection continues to be strongly associated with survival., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)
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- 2022
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5. Somatic PIK3R1 variation as a cause of vascular malformations and overgrowth.
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Cottrell CE, Bender NR, Zimmermann MT, Heusel JW, Corliss M, Evenson MJ, Magrini V, Corsmeier DJ, Avenarius M, Dudley JN, Johnston JJ, Lindhurst MJ, Vigh-Conrad K, Davies OMT, Coughlin CC, Frieden IJ, Tollefson M, Zaenglein AL, Ciliberto H, Tosi LL, Semple RK, Biesecker LG, and Drolet BA
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- Humans, Mutation, Phosphatidylinositol 3-Kinases genetics, Signal Transduction, Class Ia Phosphatidylinositol 3-Kinase genetics, Limb Deformities, Congenital, Vascular Malformations genetics
- Abstract
Purpose: Somatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1., Methods: Affected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity., Results: The phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone., Conclusion: Somatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway., (© 2021. The Author(s).)
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- 2021
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6. A Next-Generation Sequencing Test for Severe Congenital Neutropenia: Utility in a Broader Clinicopathologic Spectrum of Disease.
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McNulty SN, Evenson MJ, Riley M, Yoest JM, Corliss MM, Heusel JW, Duncavage EJ, and Pfeifer JD
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- Chromosomes, Human, Pair 7 genetics, Cohort Studies, Gene Dosage, Genome, Human, Humans, Mutation genetics, Neutropenia genetics, Neutropenia pathology, Reproducibility of Results, Congenital Bone Marrow Failure Syndromes genetics, Congenital Bone Marrow Failure Syndromes pathology, High-Throughput Nucleotide Sequencing, Neutropenia congenital
- Abstract
Severe congenital neutropenia (SCN) is a collection of diverse disorders characterized by chronically low absolute neutrophil count in the peripheral blood, increased susceptibility to infection, and a significant predisposition to the development of myeloid malignancies. SCN can be acquired or inherited. Inherited forms have been linked to variants in a group of diverse genes involved in the neutrophil-differentiation process. Variants that promote resistance to treatment have also been identified. Thus, genetic testing is important for the diagnosis, prognosis, and management of SCN. Herein we describe clinically validated assay developed for assessing patients with suspected SCN. The assay is performed from a whole-exome backbone. Variants are called across all coding exons, and results are filtered to focus on 48 genes that are clinically relevant to SCN. Validation results indicated 100% analytical sensitivity and specificity for the detection of constitutional variants among the 48 reportable genes. To date, 34 individuals have been referred for testing (age range: birth to 67 years). Several pathogenic and likely pathogenic variants have been identified, including one in a patient with late-onset disease. The pattern of cases referred for testing suggests that this assay has clinical utility in a broader spectrum of patients beyond those in the pediatric population who have classic early-onset symptoms characteristic of SCN., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)
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- 2021
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7. Identification of challenges and a framework for implementation of the AMP/ASCO/CAP classification guidelines for reporting somatic variants.
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Parikh BA, Love-Gregory L, Duncavage EJ, and Heusel JW
- Abstract
Objectives: In 2017, AMP, ASCO and CAP jointly published the first formalized classification system for the interpretation and reporting of sequence variants in cancer. The challenges of incorporating new variant interpretation guidelines into existing, validated workflows have likely hampered adoption and implementation in labs with classification methods in place. Ambiguity in assigning clinical significance across guidelines is grounded in differential weighting of evidence used in variant assessment. Therefore, we undertook an internal process-improvement exercise to correlate the two classification schemes using historical laboratory data., Design and Methods: Existing clinical variant assignments from 40 consecutive oncology cases comprising 150 somatic variants were re-assessed according to the 2017 AMP/ASCO/CAP scheme. Approximately 50% of these were cancers of the gynecologic tract., Results: Our laboratory-developed (GPS) classifications for 'actionable' variants and variants of uncertain clinical significance mapped consistently with the AMP/ASCO/CAP Tiers I-III. The majority of Level 1 variants were reclassified to Tier I (21/25; 84%) while all Level 2 and Level 4 variants were assigned to Tier II (9/9; 100%) and Tier III (17/17; 100%), respectively. The greatest variability was seen for GPS Level 3 variants, which was strongly influenced by TP53 interpretations. Ultimately, we found that most GPS Level 3 variants were classified as Tier III (77/99; 77.8%)., Conclusions: Our internally developed 5-level classifications mapped consistently with the proposed AMP/ASCO/CAP 4-Tiered system. As a result of this analysis, we can provide a framework for other labs considering a similar transition to the 2017 AMP/ASCO/CAP guidelines and a rationale for explaining specific discrepancies., Competing Interests: No conflicts of interest are declared by the authors., (© 2020 The Authors.)
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- 2020
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8. Beyond Panel-Based Testing: Exome Analysis Increases Sensitivity for Diagnosis of Genetic Kidney Disease.
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Wilson PC, Love-Gregory L, Corliss M, McNulty S, Heusel JW, and Gaut JP
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- Apolipoprotein L1, Homozygote, Humans, Retrospective Studies, Sequence Deletion, Exome genetics, Kidney Diseases
- Abstract
Background: Next-generation sequencing (NGS) is a useful tool for evaluating patients with suspected genetic kidney disease. Clinical practice relies on the use of targeted gene panels that are ordered based on patient presentation. We compare the diagnostic yield of clinical panel-based testing to exome analysis., Methods: In total, 324 consecutive patients underwent physician-ordered, panel-based NGS testing between December 2014 and October 2018. Gene panels were available for four clinical phenotypes, including atypical hemolytic uremic syndrome ( n =224), nephrotic syndrome ( n =56), cystic kidney disease ( n =26), and Alport syndrome ( n =13). Variants were analyzed and clinical reports were signed out by a pathologist or clinical geneticist at the time of testing. Subsequently, all patients underwent retrospective exome analysis to detect additional clinically significant variants in kidney disease genes that were not analyzed as part of the initial clinical gene panel. Resulting variants were classified according to the American College of Medical Genetics and Genomics 2015 guidelines., Results: In the initial physician-ordered gene panels, we identified clinically significant pathogenic or likely pathogenic variants in 13% of patients ( n =42/324). CFHR3-CFHR1 homozygous deletion was detected in an additional 13 patients with aHUS without a pathogenic or likely pathogenic variant. Diagnostic yield of the initial physician-ordered gene panel was 20% and varied between groups. Retrospective exome analysis identified 18 patients with a previously unknown pathogenic or likely pathogenic variant in a kidney disease gene and eight patients with a high-risk APOL1 genotype. Overall, retrospective exome analysis increased the diagnostic yield of panel-based testing from 20% to 30%., Conclusions: These results highlight the importance of a broad and collaborative approach between the clinical laboratory and their physician clients that employs additional analysis when a targeted panel of kidney disease-causing genes does not return a clinically meaningful result., Competing Interests: J. Gaut reports personal fees from BioLegend, grants from National Institutes of Health, and grants from Mid America Transplant Foundation, outside the submitted work. All remaining authors have nothing to disclose., (Copyright © 2020 by the American Society of Nephrology.)
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- 2020
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9. Diagnostic Utility of Next-Generation Sequencing for Disorders of Somatic Mosaicism: A Five-Year Cumulative Cohort.
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McNulty SN, Evenson MJ, Corliss MM, Love-Gregory LD, Schroeder MC, Cao Y, Lee YS, Drolet BA, Neidich JA, Cottrell CE, and Heusel JW
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- Biopsy, Cohort Studies, Humans, Genetic Testing methods, High-Throughput Nucleotide Sequencing methods, Mosaicism
- Abstract
Disorders of somatic mosaicism (DoSM) are a diverse group of syndromic and non-syndromic conditions caused by mosaic variants in genes that regulate cell survival and proliferation. Despite overlap in gene space and technical requirements, few clinical labs specialize in DoSM compared to oncology. We adapted a high-sensitivity next-generation sequencing cancer assay for DoSM in 2014. Some 343 individuals have been tested over the past 5 years, 58% of which had pathogenic and likely pathogenic (P/LP) findings, for a total of 206 P/LP variants in 22 genes. Parameters associated with the high diagnostic yield were: (1) deep sequencing (∼2,000× coverage), (2) a broad gene set, and (3) testing affected tissues. Fresh and formalin-fixed paraffin embedded tissues performed equivalently for identification of P/LP variants (62% and 71% of individuals, respectively). Comparing cultured fibroblasts to skin biopsies suggested that culturing might boost the allelic fraction of variants that confer a growth advantage, specifically gain-of-function variants in PIK3CA. Buccal swabs showed high diagnostic sensitivity in case subjects where disease phenotypes manifested in the head or brain. Peripheral blood was useful as an unaffected comparator tissue to determine somatic versus constitutional origin but had poor diagnostic sensitivity. Descriptions of all tested individuals, specimens, and P/LP variants included in this cohort are available to further the study of the DoSM population., (Copyright © 2019. Published by Elsevier Inc.)
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- 2019
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10. Optimization of Population Frequency Cutoffs for Filtering Common Germline Polymorphisms from Tumor-Only Next-Generation Sequencing Data.
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McNulty SN, Parikh BA, Duncavage EJ, Heusel JW, and Pfeifer JD
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- Computational Biology methods, Gene Frequency, Genome, Human, High-Throughput Nucleotide Sequencing methods, Humans, Neoplasms epidemiology, Neoplasms genetics, United States epidemiology, Computational Biology standards, Genetic Predisposition to Disease, Genetics, Population standards, Germ-Line Mutation, High-Throughput Nucleotide Sequencing standards, Neoplasms diagnosis, Polymorphism, Genetic
- Abstract
Clinical next-generation sequencing assays are often run on tumor specimens without a matched normal specimen, which complicates the differentiation of germline from somatic variants. In tumor-only testing, population data are often used to infer germline status, though no consensus exists on the exact population frequency (PF) cutoff above which a variant should be considered likely germline. In this study, five population databases plus the Catalog of Somatic Mutations in Cancer were used to demonstrate the impact of changing the PF cutoff on assignment of variants as germline versus somatic. The 1% to 2% PF cutoffs widely used in bioinformatic pipelines resulted in high sensitivity for classification of somatic variants, but unnecessarily reduced sensitivity for germline variants. Using optimized PF cutoffs, the source of variants in The Cancer Genome Atlas (TCGA) data could be predicted with >95% accuracy. Further exploration of four TCGA cancer data sets indicated that the optimal cutoff is influenced by both cancer type and the assay region of interest. Comparing TCGA data to data generated from a clinical, hybridization capture test (approximately 615 kb capture space) showed that PF cutoffs may not be transferable between assays, even when the gene set is held constant. Thus, filtering approaches need to be carefully designed and optimized, and should be assay-specific to support tumor-only testing until tumor-normal testing becomes routine in the clinical setting., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)
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- 2019
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11. A Multimodality Approach to Assessing Factor I Genetic Variants in Atypical Hemolytic Uremic Syndrome.
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Java A, Pozzi N, Love-Gregory LD, Heusel JW, Sung YJ, Hu Z, Bertram P, Liszewski MK, Cline LM, Ren Z, and Atkinson JP
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- 2019
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12. Beyond sequence variation: assessment of copy number variation in adult glioblastoma through targeted tumor somatic profiling.
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McNulty SN, Cottrell CE, Vigh-Conrad KA, Carter JH, Heusel JW, Ansstas G, and Dahiya S
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- Adult, Aged, Aged, 80 and over, Brain Neoplasms pathology, ErbB Receptors genetics, Female, Gene Amplification, Gene Expression Regulation, Neoplastic, Glioblastoma pathology, High-Throughput Nucleotide Sequencing, Humans, Male, Middle Aged, Prognosis, Brain Neoplasms genetics, DNA Copy Number Variations, Glioblastoma genetics, Mutation
- Abstract
Glioblastoma is the most common primary malignancy of the adult central nervous system. Gliomagenesis involves a complex range of alterations, including sequence changes, copy number variations (CNVs), and epigenetic modifications, that have clinical implications for disease classification and prognosis. Thus, multiple testing modalities are required to support a complete diagnostic workup. The goal of this study was to streamline the multipart workflow by predicting both sequence changes and CNVs (specifically EGFR amplifications) from a single next-generation sequencing (NGS) test. Eighty-six primary and secondary glioblastomas were submitted for clinical NGS to report sequence variants from a concise panel of cancer-relevant genes. Most specimens underwent concomitant testing by methylation-specific polymerase chain reaction, immunohistochemistry, and fluorescence in situ hybridization. Using data generated during the course of clinical testing, we found that NGS-based variant predictions were concordant with immunohistochemistry and fluorescence in situ hybridization for IDH mutation and EGFR amplification status, respectively. We also noted that EGFR amplifications correlated with polysomy of chromosome 7, 19, and 20, and loss of PTEN and CDKN2A. EGFR-unamplified cases had lower rates of chromosome 7 polysomy, and PTEN and CDKN2A loss, but more CNVs overall. TP53, NF1, ATRX, and PDGFRA mutations were nearly exclusive to specimens without EGFR amplification. EGFR amplification was not associated with longer progression-free survival in this cohort, but amplifications were enriched in a group with slightly longer overall survival despite radiographic evidence of disease progression. Further study is needed to explore the mechanisms responsible for noted patterns of co-occurring variants and to correlate them with specific clinical outcomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)
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- 2019
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13. Clinical Targeted Next-Generation Sequencing Shows Increased Mutational Load in Endometrioid-type Endometrial Adenocarcinoma With Deficient DNA Mismatch Repair.
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Lee PJ, McNulty S, Duncavage EJ, Heusel JW, and Hagemann IS
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- Adult, Aged, Aged, 80 and over, Carcinoma, Endometrioid pathology, DNA-Binding Proteins genetics, Endometrial Neoplasms pathology, Female, High-Throughput Nucleotide Sequencing, Humans, Middle Aged, Mismatch Repair Endonuclease PMS2 genetics, MutL Protein Homolog 1 genetics, MutS Homolog 2 Protein genetics, Carcinoma, Endometrioid genetics, DNA Mismatch Repair genetics, Endometrial Neoplasms genetics, Microsatellite Instability, Mutation
- Abstract
A subset of endometrial adenocarcinomas (EACs) exhibit microsatellite instability and have deficient DNA mismatch repair (dMMR). The overall aim of the study was to compare the spectrum of mutations in endometrioid-type EAC with and without dMMR by using a clinically validated next-generation sequencing assay. We retrospectively identified 19 EACs with known mismatch repair status that had undergone targeted sequencing of a panel of cancer-related genes. The mismatch repair status was ascertained by immunohistochemistry against MLH1, PMS2, MSH2, and MSH6 mismatch proteins. Somatic mutations in EAC with dMMR were compared against those in cases with proficient MMR (pMMR). The dMMR EAC showed a normalized mean of 66.6 mutations/Mb per case compared with pMMR EAC with a mean of 26.2 (P<0.05). The most commonly mutated genes were PTEN (89% of dMMR, 50% of pMMR), PIK3CA (67% vs. 40%), ATM (89% vs. 40%), and FLT3 (67% vs. 50%). The transition/transversion ratio was 4.7 versus 2.8 for the dMMR and pMMR cohorts, respectively (P<0.05). Copy number variant analysis did not demonstrate significant differences between the dMMR and pMMR cohorts and was not correlated with histologic grade of EAC. In conclusion, tumorigenesis of EAC in the context of dMMR demonstrated heavier mutational burdens and higher transition/transversion ratio. The spectrum of genetic alterations can potentially help identify cases with microsatellite instability phenotype using next-generation sequencing data from a targeted cancer gene panel.
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- 2018
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14. Discriminating a common somatic ASXL1 mutation (c.1934dup; p.G646Wfs*12) from artifact in myeloid malignancies using NGS.
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Alberti MO, Srivatsan SN, Shao J, McNulty SN, Chang GS, Miller CA, Dunlap JB, Yang F, Press RD, Gao Q, Ding L, Heusel JW, Duncavage EJ, and Walter MJ
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- Artifacts, Diagnosis, Differential, Hematologic Neoplasms genetics, High-Throughput Nucleotide Sequencing methods, Humans, Molecular Diagnostic Techniques, Myeloproliferative Disorders genetics, Prognosis, Biomarkers, Tumor genetics, Gene Duplication, Hematologic Neoplasms diagnosis, High-Throughput Nucleotide Sequencing standards, Mutation, Myeloproliferative Disorders diagnosis, Repressor Proteins genetics
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- 2018
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15. Acute graft-versus-host disease following lung transplantation in a patient with a novel TERT mutation.
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Brestoff JR, Vessoni AT, Brenner KA, Uy GL, DiPersio JF, Blinder M, Witt CA, Byers DE, Hachem RR, Truclock EP, Early DS, Anadkat MJ, Musiek A, Javidan-Nejad C, Balfe DM, Rosman IS, Liu C, Zhang L, Despotis GJ, Ruzinova MB, Sehn JK, Amarillo I, Heusel JW, Swat W, Kim BS, Wartman LD, Yusen RD, and Batista LFZ
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- Acute Disease, Fatal Outcome, Female, Graft vs Host Disease pathology, Humans, Mutation, Pulmonary Fibrosis surgery, Telomerase metabolism, Graft vs Host Disease etiology, Lung Transplantation adverse effects, Pulmonary Fibrosis genetics, Telomerase genetics
- Abstract
Familial pulmonary fibrosis is associated with loss-of-function mutations in telomerase reverse transcriptase ( TERT ) and short telomeres. Interstitial lung diseases have become the leading indication for lung transplantation in the USA, and recent data indicate that pathogenic mutations in telomerase may cause unfavourable outcomes following lung transplantation. Although a rare occurrence, solid organ transplant recipients who develop acute graft-versus-host disease (GVHD) have very poor survival. This case report describes the detection of a novel mutation in TERT in a patient who had lung transplantation for familial pulmonary fibrosis and died from complications of acute GVHD., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)
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- 2018
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16. Natural Killer Cell Recruitment to the Lung During Influenza A Virus Infection Is Dependent on CXCR3, CCR5, and Virus Exposure Dose.
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Carlin LE, Hemann EA, Zacharias ZR, Heusel JW, and Legge KL
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- Animals, Chemotaxis, Leukocyte immunology, Influenza A virus, Lung immunology, Lung virology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Killer Cells, Natural immunology, Orthomyxoviridae Infections immunology, Orthomyxoviridae Infections virology, Receptors, CCR5 immunology, Receptors, CXCR3 immunology
- Abstract
Natural killer (NK) cells are vital components of the antiviral immune response, but their contributions in defense against influenza A virus (IAV) are not well understood. To better understand NK cell responses during IAV infections, we examined the magnitude, kinetics, and contribution of NK cells to immunity and protection during high- and low-dose IAV infections. Herein, we demonstrate an increased accumulation of NK cells in the lung in high-dose vs. low-dose infections. In part, this increase is due to the local proliferation of pulmonary NK cells. However, the majority of NK cell accumulation within the lungs and airways during an IAV infection is due to recruitment that is partially dependent upon CXCR3 and CCR5, respectively. Therefore, altogether, our results demonstrate that NK cells are actively recruited to the lungs and airways during IAV infection and that the magnitude of the recruitment may relate to the inflammatory environment found within the tissues during high- and low-dose IAV infections.
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- 2018
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17. Routine use of clinical exome-based next-generation sequencing for evaluation of patients with thrombotic microangiopathies.
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Gaut JP, Jain S, Pfeifer JD, Vigh-Conrad KA, Corliss M, Sharma MK, Heusel JW, and Cottrell CE
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- Adult, Aged, Case-Control Studies, Cohort Studies, Exome, Female, Humans, Male, Middle Aged, High-Throughput Nucleotide Sequencing methods, Thrombotic Microangiopathies diagnosis, Thrombotic Microangiopathies genetics
- Abstract
Next-generation sequencing is increasingly used for clinical evaluation of patients presenting with thrombotic microangiopathies because it allows for simultaneous interrogation of multiple complement and coagulation pathway genes known to be associated with disease. However, the diagnostic yield is undefined in routine clinical practice. Historic studies relied on case-control cohorts, did not apply current guidelines for variant pathogenicity assessment, and used targeted gene enrichment combined with next-generation sequencing. A clinically enhanced exome, targeting ~54 Mb, was sequenced for 73 patients. Variant analysis and interpretation were performed on genes with biological relevance in thrombotic microangiopathy (C3,CD46, CFB, CFH, CFI, DGKE, and THBD). CFHR3-CFHR1 deletion status was also assessed using multiplex ligation-dependent probe amplification. Variants were classified using American College of Medical Genetics and Genomics guidelines. We identified 5 unique novel and 14 unique rare variants in 25% (18/73) of patients, including a total of 5 pathogenic, 4 likely pathogenic, and 15 variants of uncertain clinical significance. Nine patients had homozygous deletions in CFHR3-CFHR1. The diagnostic yield, defined as the presence of a pathogenic variant, likely pathogenic variant or homozygous deletion of CFHR3-CFHR1, was 25% for all patients tested. Variants of uncertain clinical significance were identified in 21% (15/73) of patients.These results illustrate the expected diagnositic yield in the setting of thrombotic microangiopathies through the application of standardized variant interpretation, and highlight the utility of such an approach. Sequencing a clinically enhanced exome to enable targeted, disease-specific variant analysis is a viable approach. The moderate rate of variants of uncertain clinical significance highlights the paucity of data surrounding the variants in our cohort and illustrates the need for expanded variant curation resources to aid in thrombotic microangiopathy-related disease variant classification.
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- 2017
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18. FGFR2 amplification in colorectal adenocarcinoma.
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Carter JH, Cottrell CE, McNulty SN, Vigh-Conrad KA, Lamp S, Heusel JW, and Duncavage EJ
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- Adenocarcinoma genetics, Cell Line, Tumor, Colorectal Neoplasms genetics, DNA Copy Number Variations genetics, Female, Gene Amplification genetics, High-Throughput Nucleotide Sequencing, Humans, Middle Aged, Receptor, Fibroblast Growth Factor, Type 2 metabolism, Stomach Neoplasms genetics, Adenomatous Polyposis Coli genetics, Receptor, Fibroblast Growth Factor, Type 2 genetics
- Abstract
FGFR2 is recurrently amplified in 5% of gastric cancers and 1%-4% of breast cancers; however, this molecular alteration has never been reported in a primary colorectal cancer specimen. Preclinical studies indicate that several FGFR tyrosine-kinase inhibitors (TKIs), such as AZD4547, have in vitro activity against the FGFR2 -amplified colorectal cell line, NCI-H716. The efficacy of these inhibitors is currently under investigation in clinical trials for breast and gastric cancer. Thus, better characterizing colorectal tumors for FGFR2 amplification could identify a subset of patients who may benefit from FGFR TKI therapies. Here, we describe a novel FGFR2 amplification identified by clinical next-generation sequencing in a primary colorectal cancer. Further characterization of the tumor by immunohistochemistry showed neuroendocrine differentiation, similar to the reported properties of the NCI-H716 cell line. These findings demonstrate that the spectrum of potentially clinically actionable mutations detected by targeted clinical sequencing panels is not limited to only single-nucleotide polymorphisms and insertions/deletions but also to copy-number alterations., (© 2017 Carter et al.; Published by Cold Spring Harbor Laboratory Press.)
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- 2017
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19. Targeted Next-Generation Sequencing in Molecular Subtyping of Lower-Grade Diffuse Gliomas: Application of the World Health Organization's 2016 Revised Criteria for Central Nervous System Tumors.
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Carter JH, McNulty SN, Cimino PJ, Cottrell CE, Heusel JW, Vigh-Conrad KA, and Duncavage EJ
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- Adolescent, Adult, Aged, Child, Child, Preschool, Computational Biology methods, DNA Copy Number Variations, Disease Management, Female, Humans, Male, Middle Aged, Mutation, Neoplasm Grading, Polymorphism, Single Nucleotide, Reproducibility of Results, Workflow, Young Adult, Biomarkers, Tumor, Brain Neoplasms diagnosis, Brain Neoplasms genetics, Glioma diagnosis, Glioma genetics, High-Throughput Nucleotide Sequencing methods
- Abstract
The 2007 World Health Organization Classification of Tumours of the Central Nervous System classifies lower-grade gliomas [LGGs (grades II to III diffuse gliomas)] morphologically as astrocytomas or oligodendrogliomas, and tumors with unclear ambiguous morphology as oligoastrocytomas. The World Health Organization's newly released (2016) classification incorporates molecular data. A single, targeted next-generation sequencing (NGS) panel was used for detecting single-nucleotide variation and copy number variation in 50 LGG cases originally classified using the 2007 criteria, including 36 oligoastrocytomas, 11 oligodendrogliomas, 2 astrocytomas, and 1 LGG not otherwise specified. NGS results were compared with those from IHC analysis and fluorescence in situ hybridization to assess concordance and to categorize the tumors according to the 2016 criteria. NGS results were concordant with those from IHC analysis in all cases. In 3 cases, NGS was superior to fluorescence in situ hybridization in distinguishing segmental chromosomal losses from whole-arm deletions. The NGS approach was effective in reclassifying 36 oligoastrocytomas as 30 astrocytomas (20 IDH1/2 mutant and 10 IDH1/2 wild type) and 6 oligodendrogliomas, and 1 oligodendroglioma as an astrocytoma (IDH1/2 mutant). Here we show that a single, targeted NGS assay can serve as the sole testing modality for categorizing LGG according to the World Health Organization's 2016 diagnostic scheme. This modality affords greater accuracy and efficiency while reducing specimen tissue requirements compared with multimodal approaches., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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20. Concurrent MPL W515L and Y591D mutations in a patient with myelofibrosis.
- Author
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Rashidi A, Heusel JW, and Oh ST
- Subjects
- Aged, Amino Acid Substitution, Humans, Male, Primary Myelofibrosis blood, Receptors, Thrombopoietin metabolism, Mutation, Missense, Primary Myelofibrosis genetics, Receptors, Thrombopoietin genetics
- Published
- 2016
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21. Extrinsic allospecific signals of hematopoietic origin dictate iNKT cell lineage-fate decisions during development.
- Author
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Strong BSI, Newkold TJ, Lee AE, Turner LE, Alhajjat AM, Heusel JW, and Shaaban AF
- Subjects
- Animals, Cell Lineage, Mice, Inbred BALB C, Mice, Inbred C57BL, NK Cell Lectin-Like Receptor Subfamily A metabolism, Natural Killer T-Cells transplantation, Transplantation Tolerance, Natural Killer T-Cells physiology
- Abstract
Invariant NKT (iNKT) cells are critical to the maintenance of tolerance toward alloantigens encountered during postnatal life pointing to the existence of a process for self-education. However, the impact of developmentally encountered alloantigens in shaping the phenotype and function of iNKT cells has not been described. To better understand this process, the current report examined naïve iNKT cells as they matured in an allogeneic environment. Following the prenatal transfer of fetal hematopoietic cells between age-matched allogeneic murine fetuses, cell-extrinsic signals appeared to dictate allospecific patterns of Ly49 receptor expression and lineage diversity in developing iNKT cells. Regulation for this process arose from cells of hematopoietic origin requiring only rare exposure to facilitate broad changes in developing iNKT cells. These findings highlight surprisingly asymmetric allospecific alterations in iNKT cells as they develop and mature in an allogeneic environment and establish a new paradigm for study of the self-education of iNKT cells.
- Published
- 2016
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22. Prenatal Allogeneic Tolerance in Mice Remains Stable Despite Potent Viral Immune Activation.
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Strong BS, Ryken KO, Lee AE, Turner LE, Wadhwani RK, Newkold TJ, Alhajjat AM, Heusel JW, and Shaaban AF
- Subjects
- Allografts, Animals, Female, Fetal Diseases immunology, Fetal Diseases therapy, Mice, Pregnancy, Hematopoietic Stem Cell Transplantation, Transplantation Chimera immunology, Transplantation Tolerance
- Abstract
Transplanting stem cells before birth offers an unparalleled opportunity to initiate corrective treatment for numerous childhood diseases with minimal or no host conditioning. Although long-term engraftment has been demonstrated following in utero hematopoietic cellular transplantation during immune quiescence, it is unclear if prenatal tolerance becomes unstable with immune activation such as during a viral syndrome. Using a murine model of in utero hematopoietic cellular transplantation, the impact of an infection with lymphocytic choriomeningitis virus on prenatal allospecific tolerance was examined. The findings in this report illustrate that established mechanisms of donor-specific tolerance are strained during potent immune activation. Specifically, a transient reversal in the anergy of alloreactive lymphocytes is seen in parallel with the global immune response toward the virus. However, these changes return to baseline following resolution of the infection. Importantly, prenatal engraftment remains stable during and after immune activation. Collectively, these findings illustrate the robust nature of allospecific tolerance in prenatal mixed chimerism compared with models of postnatal chimerism and provides additional support for the prenatal approach to the treatment of congenital benign cellular disease., (Copyright © 2015 by The American Association of Immunologists, Inc.)
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- 2015
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23. Identification of Medically Actionable Secondary Findings in the 1000 Genomes.
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Olfson E, Cottrell CE, Davidson NO, Gurnett CA, Heusel JW, Stitziel NO, Chen LS, Hartz S, Nagarajan R, Saccone NL, and Bierut LJ
- Subjects
- Databases, Genetic, Genetic Predisposition to Disease, Genetic Testing, Humans, Mutation, Genetic Variation, Genome, Human, Genomics methods
- Abstract
The American College of Medical Genetics and Genomics (ACMG) recommends that clinical sequencing laboratories return secondary findings in 56 genes associated with medically actionable conditions. Our goal was to apply a systematic, stringent approach consistent with clinical standards to estimate the prevalence of pathogenic variants associated with such conditions using a diverse sequencing reference sample. Candidate variants in the 56 ACMG genes were selected from Phase 1 of the 1000 Genomes dataset, which contains sequencing information on 1,092 unrelated individuals from across the world. These variants were filtered using the Human Gene Mutation Database (HGMD) Professional version and defined parameters, appraised through literature review, and examined by a clinical laboratory specialist and expert physician. Over 70,000 genetic variants were extracted from the 56 genes, and filtering identified 237 variants annotated as disease causing by HGMD Professional. Literature review and expert evaluation determined that 7 of these variants were pathogenic or likely pathogenic. Furthermore, 5 additional truncating variants not listed as disease causing in HGMD Professional were identified as likely pathogenic. These 12 secondary findings are associated with diseases that could inform medical follow-up, including cancer predisposition syndromes, cardiac conditions, and familial hypercholesterolemia. The majority of the identified medically actionable findings were in individuals from the European (5/379) and Americas (4/181) ancestry groups, with fewer findings in Asian (2/286) and African (1/246) ancestry groups. Our results suggest that medically relevant secondary findings can be identified in approximately 1% (12/1092) of individuals in a diverse reference sample. As clinical sequencing laboratories continue to implement the ACMG recommendations, our results highlight that at least a small number of potentially important secondary findings can be selected for return. Our results also confirm that understudied populations will not reap proportionate benefits of genomic medicine, highlighting the need for continued research efforts on genetic diseases in these populations.
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- 2015
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24. Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development.
- Author
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Alhajjat AM, Strong BS, Lee AE, Turner LE, Wadhwani RK, Ortaldo JR, Heusel JW, and Shaaban AF
- Subjects
- Adoptive Transfer, Animals, Bone Marrow Transplantation, Clonal Anergy genetics, Clonal Anergy immunology, Graft Rejection immunology, H-2 Antigens immunology, Homeostasis, Immunophenotyping, Killer Cells, Natural cytology, Mice, Models, Animal, Phenotype, Transplantation Chimera, Immune Tolerance, Isoantigens immunology, Killer Cells, Natural immunology, Killer Cells, Natural metabolism, Lymphocyte Activation, Receptors, Immunologic metabolism
- Abstract
Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or immunity. Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell-allospecific education., (Copyright © 2015 by The American Association of Immunologists, Inc.)
- Published
- 2015
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25. An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge.
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Brownstein CA, Beggs AH, Homer N, Merriman B, Yu TW, Flannery KC, DeChene ET, Towne MC, Savage SK, Price EN, Holm IA, Luquette LJ, Lyon E, Majzoub J, Neupert P, McCallie D Jr, Szolovits P, Willard HF, Mendelsohn NJ, Temme R, Finkel RS, Yum SW, Medne L, Sunyaev SR, Adzhubey I, Cassa CA, de Bakker PI, Duzkale H, Dworzyński P, Fairbrother W, Francioli L, Funke BH, Giovanni MA, Handsaker RE, Lage K, Lebo MS, Lek M, Leshchiner I, MacArthur DG, McLaughlin HM, Murray MF, Pers TH, Polak PP, Raychaudhuri S, Rehm HL, Soemedi R, Stitziel NO, Vestecka S, Supper J, Gugenmus C, Klocke B, Hahn A, Schubach M, Menzel M, Biskup S, Freisinger P, Deng M, Braun M, Perner S, Smith RJ, Andorf JL, Huang J, Ryckman K, Sheffield VC, Stone EM, Bair T, Black-Ziegelbein EA, Braun TA, Darbro B, DeLuca AP, Kolbe DL, Scheetz TE, Shearer AE, Sompallae R, Wang K, Bassuk AG, Edens E, Mathews K, Moore SA, Shchelochkov OA, Trapane P, Bossler A, Campbell CA, Heusel JW, Kwitek A, Maga T, Panzer K, Wassink T, Van Daele D, Azaiez H, Booth K, Meyer N, Segal MM, Williams MS, Tromp G, White P, Corsmeier D, Fitzgerald-Butt S, Herman G, Lamb-Thrush D, McBride KL, Newsom D, Pierson CR, Rakowsky AT, Maver A, Lovrečić L, Palandačić A, Peterlin B, Torkamani A, Wedell A, Huss M, Alexeyenko A, Lindvall JM, Magnusson M, Nilsson D, Stranneheim H, Taylan F, Gilissen C, Hoischen A, van Bon B, Yntema H, Nelen M, Zhang W, Sager J, Zhang L, Blair K, Kural D, Cariaso M, Lennon GG, Javed A, Agrawal S, Ng PC, Sandhu KS, Krishna S, Veeramachaneni V, Isakov O, Halperin E, Friedman E, Shomron N, Glusman G, Roach JC, Caballero J, Cox HC, Mauldin D, Ament SA, Rowen L, Richards DR, San Lucas FA, Gonzalez-Garay ML, Caskey CT, Bai Y, Huang Y, Fang F, Zhang Y, Wang Z, Barrera J, Garcia-Lobo JM, González-Lamuño D, Llorca J, Rodriguez MC, Varela I, Reese MG, De La Vega FM, Kiruluta E, Cargill M, Hart RK, Sorenson JM, Lyon GJ, Stevenson DA, Bray BE, Moore BM, Eilbeck K, Yandell M, Zhao H, Hou L, Chen X, Yan X, Chen M, Li C, Yang C, Gunel M, Li P, Kong Y, Alexander AC, Albertyn ZI, Boycott KM, Bulman DE, Gordon PM, Innes AM, Knoppers BM, Majewski J, Marshall CR, Parboosingh JS, Sawyer SL, Samuels ME, Schwartzentruber J, Kohane IS, and Margulies DM
- Subjects
- Child, Female, Financing, Organized, Genetic Testing economics, Genetic Testing standards, Genomics economics, Genomics standards, Heart Defects, Congenital diagnosis, Heart Defects, Congenital genetics, Humans, Male, Myopathies, Structural, Congenital diagnosis, Myopathies, Structural, Congenital genetics, Sequence Analysis, DNA economics, Sequence Analysis, DNA standards, Databases, Genetic standards, Genetic Testing methods, Genomics methods, Peer Review, Research, Sequence Analysis, DNA methods
- Abstract
Background: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance., Results: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization., Conclusions: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.
- Published
- 2014
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26. The Glycophosphatidylinositol Anchor of the MCMV Evasin, m157, Facilitates Optimal Cell Surface Expression and Ly49 Receptor Recognition.
- Author
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Carlin LE, Guseva NV, Shey MR, Ballas ZK, and Heusel JW
- Subjects
- Animals, Cytotoxicity, Immunologic, Down-Regulation, Mice, Mice, Inbred C57BL, Muromegalovirus immunology, Glycosylphosphatidylinositols metabolism, Killer Cells, Natural immunology, Muromegalovirus metabolism, NK Cell Lectin-Like Receptor Subfamily A metabolism, Viral Proteins metabolism
- Abstract
The murine cytomegalovirus-encoded protein m157 is a cognate ligand for both inhibitory and activating receptors expressed by natural killer cells. Additionally, m157 is expressed on the surface of infected cells by a glycophosphatidylinositol (GPI) anchor. Although endogenous GPI-anchored proteins are known to be ligands for the NK cell receptor, NKG2D, the contribution of the GPI anchor for viral m157 ligand function is unknown. To determine whether the GPI anchor for m157 is dispensable for m157 function, we generated m157 variants expressed as transmembrane fusion proteins and tested cells expressing transmembrane m157 for the capacity to activate cognate Ly49 receptors. We found that the GPI anchor is required for high-level cell surface expression of m157, and that the transmembrane m157 ligand retains the capacity to activate reporter cells and NK cells expressing Ly49H, as well as Ly49I(129) reporter cells, but with reduced potency. Importantly, target cells expressing the transmembrane form of m157 were killed less efficiently and failed to mediate Ly49H receptor downregulation on fresh NK cells compared to targets expressing GPI-anchored m157. Taken together, these results show that the GPI anchor for m157 facilitates robust cell surface expression, and that NK cells are sensitive to the altered cell surface expression of this potent viral evasin.
- Published
- 2013
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27. Role of NK cell subsets in organ-specific murine melanoma metastasis.
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Ballas ZK, Buchta CM, Rosean TR, Heusel JW, and Shey MR
- Subjects
- Animals, Cell Line, Tumor, Killer Cells, Natural immunology, Liver Neoplasms immunology, Liver Neoplasms secondary, Lung Neoplasms immunology, Lung Neoplasms secondary, Male, Melanoma, Mice, Killer Cells, Natural metabolism, Melanoma, Experimental complications, Melanoma, Experimental immunology
- Abstract
Tumor metastasis plays a major role in the morbidity and mortality of cancer patients. Among solid tumors that undergo metastasis, there is often a predilection to metastasize to a particular organ with, for example, prostate cancer preferentially metastasizing to bones and colon cancer preferentially metastasizing to the liver. Although many factors are thought to be important in establishing permissiveness for metastasis, the reasons for organ-specific predilection of each tumor are not understood. Using a B16 murine melanoma model, we tested the hypothesis that organ-specific NK cell subsets play a critical role in organ-specific metastasis of this tumor. Melanoma cells, given intravenously, readily colonized the lungs but not the liver. NK cell depletion (either iatrogenically or by using genetically targeted mice) resulted in substantial hepatic metastasis. Analysis of NK cell subsets, defined by the differential expression of a combination of CD27 and CD11b, indicated a significant difference in the distribution of NK cell subsets in the lung and liver with the mature subset being dominant in the lung and the immature subset being dominant in the liver. Several experimental approaches, including adoptive transfer, clearly indicated that the immature hepatic NK cell subset, CD27+ CD11b-, was protective against liver metastasis; this subset mediated its protection by a perforin-dependent cytotoxic mechanism. In contrast, the more mature NK cell subsets were more efficient at reducing pulmonary tumor load. These data indicate that organ-specific immune responses may play a pivotal role in determining the permissiveness of a given organ for the establishment of a metastatic niche.
- Published
- 2013
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28. Perforin plays an unexpected role in regulating T-cell contraction during prolonged Listeria monocytogenes infection.
- Author
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Schmidt NW, Khanolkar A, Hancox L, Heusel JW, and Harty JT
- Subjects
- Animals, CD4-Positive T-Lymphocytes microbiology, CD8-Positive T-Lymphocytes microbiology, Cell Proliferation, Flow Cytometry, Immunologic Memory immunology, Listeriosis microbiology, Mice, Mice, Inbred C57BL, Mice, Knockout, Specific Pathogen-Free Organisms, Tumor Necrosis Factor-alpha immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Listeria monocytogenes immunology, Listeriosis immunology, Perforin immunology
- Abstract
After infection or vaccination, antigen-specific T cells proliferate then contract in numbers to a memory set point. T-cell contraction is observed after both acute and prolonged infections although it is unknown if contraction is regulated similarly in both scenarios. Here, we show that contraction of antigen-specific CD8(+) and CD4(+) T cells is markedly reduced in TNF/perforin-double deficient (DKO) mice responding to attenuated Listeria monocytogenes infection. Reduced contraction in DKO mice was associated with delayed clearance of infection and sustained T-cell proliferation during the normal contraction interval. Mechanistically, sustained T-cell proliferation mapped to prolonged infection in the absence of TNF; however, reduced contraction required the additional absence of perforin since T cells in mice lacking either TNF or perforin (singly deficient) underwent normal contraction. Thus, while T-cell contraction after acute infection is independent of peforin, a perforin-dependent pathway plays a previously unappreciated role to mediate contraction of antigen-specific CD8(+) and CD4(+) T cells during prolonged L. monocytogenes infection., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
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29. Glycosylation contributes to variability in expression of murine cytomegalovirus m157 and enhances stability of interaction with the NK-cell receptor Ly49H.
- Author
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Guseva NV, Fullenkamp CA, Naumann PW, Shey MR, Ballas ZK, Houtman JC, Forbes CA, Scalzo AA, and Heusel JW
- Subjects
- Animals, Cell Line, Fibroblasts immunology, Fibroblasts pathology, Glycosylation, Herpesviridae Infections genetics, Herpesviridae Infections immunology, Herpesviridae Infections virology, Killer Cells, Natural immunology, Killer Cells, Natural metabolism, Killer Cells, Natural pathology, Lymphocyte Activation genetics, Mice, Muromegalovirus pathogenicity, Mutagenesis, Site-Directed, Mutation genetics, Myeloid Cells immunology, Myeloid Cells pathology, NK Cell Lectin-Like Receptor Subfamily A immunology, NK Cell Lectin-Like Receptor Subfamily A metabolism, Protein Binding genetics, Protein Isoforms genetics, Transgenes genetics, Viral Proteins genetics, Viral Proteins immunology, Fibroblasts metabolism, Herpesviridae Infections metabolism, Muromegalovirus immunology, Myeloid Cells metabolism, Viral Proteins metabolism
- Abstract
NK cell-mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol-linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate-binding lectins, the role of glycosylation in ligand binding is unclear. Herein, we show that m157 is expressed in multiple, differentially N-glycosylated isoforms in m157-transduced or MCMV-infected cells. We used site-directed mutagenesis to express single and combinatorial asparagine (N)-to-glutamine (Q) mutations at N178, N187, N213, and N267 in myeloid and fibroblast cell lines. Progressive loss of N-linked glycans led to a significant reduction of total cellular m157 abundance, although all variably glycosylated m157 isoforms were expressed at the cell surface and retained the capacity to activate Ly49H(B6) and Ly49I(129) reporter cells and Ly49H(+) NK cells. However, the complete lack of N-linked glycans on m157 destabilized the m157-Ly49H interaction and prevented physical transfer of m157 to Ly49H-expressing cells. Thus, glycosylation on m157 enhances expression and binding to Ly49H, factors that may impact the interaction between NK cells and MCMV in vivo where receptor-ligand interactions are more limiting.
- Published
- 2010
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30. Innate immune control and regulation of influenza virus infections.
- Author
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McGill J, Heusel JW, and Legge KL
- Subjects
- Animals, Humans, Virus Replication immunology, Dendritic Cells immunology, Immunity, Innate, Influenza A virus immunology, Influenza, Human immunology, Killer Cells, Natural immunology, Macrophages, Alveolar immunology
- Abstract
Adaptive immune responses are critical for the control and clearance of influenza A virus (IAV) infection. However, in recent years, it has become increasingly apparent that innate immune cells, including natural killer cells, alveolar macrophages (aMphi), and dendritic cells (DC) are essential following IAV infection in the direct control of viral replication or in the induction and regulation of virus-specific adaptive immune responses. This review will discuss the role of these innate immune cells following IAV infection, with a particular focus on DC and their ability to induce and regulate the adaptive IAV-specific immune response.
- Published
- 2009
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31. Characterization of murine cytomegalovirus m157 from infected cells and identification of critical residues mediating recognition by the NK cell receptor Ly49H.
- Author
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Davis AH, Guseva NV, Ball BL, and Heusel JW
- Subjects
- Animals, Antibodies, Monoclonal immunology, Antigens, Ly chemistry, Cell Line, Cell Membrane immunology, Cell Membrane metabolism, Gene Expression Regulation, Viral, Glycoproteins chemistry, Glycoproteins genetics, Glycoproteins metabolism, Glycosylphosphatidylinositols metabolism, Lectins, C-Type chemistry, Mice, Models, Molecular, Muromegalovirus chemistry, Muromegalovirus genetics, Muromegalovirus metabolism, Mutation genetics, NK Cell Lectin-Like Receptor Subfamily A, Protein Binding, Protein Isoforms immunology, Protein Structure, Tertiary, Receptors, NK Cell Lectin-Like, Viral Proteins chemistry, Viral Proteins genetics, Viral Proteins metabolism, Antigens, Ly immunology, Glycoproteins immunology, Lectins, C-Type immunology, Muromegalovirus immunology, Viral Proteins immunology
- Abstract
Activated NK cells mediate potent cytolytic and secretory effector functions and are vital components of the early antiviral immune response. NK cell activities are regulated by the assortment of inhibitory receptors that recognize MHC class I ligands expressed on healthy cells and activating receptors that recognize inducible host ligands or ligands that are not well characterized. The activating Ly49H receptor of mouse NK cells is unique in that it specifically recognizes a virally encoded ligand, the m157 glycoprotein of murine CMV (MCMV). The Ly49H-m157 interaction underlies a potent resistance mechanism (Cmv1) in C57BL/6 mice and serves as an excellent model in which to understand how NK cells are specifically activated in vivo, as similar receptor systems are operative for human NK cells. For transduced cells expressing m157 in isolation and for MCMV-infected cells, we show that m157 is expressed in multiple isoforms with marked differences in abundance between infected fibroblasts (high) and macrophages (low). At the cell surface, m157 is exclusively a glycosylphosphatidylinositol-associated protein in MCMV-infected cells. Through random and site-directed mutagenesis of m157, we identify unique residues that provide for efficient cell surface expression of m157 but fail to activate Ly49H-expressing reporter cells. These m157 mutations are predicted to alter the conformation of a putative m157 interface with Ly49H, one that relies on the position of a critical alpha0 helix of m157. These findings support an emerging model for a novel interaction between this important NK cell receptor and its viral ligand.
- Published
- 2008
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32. Natural killer cells: emerging concepts in immunity to infection and implications for assessment of immunodeficiency.
- Author
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Heusel JW and Ballas ZK
- Subjects
- Animals, Humans, Interferon-gamma physiology, Killer Cells, Natural physiology, Models, Immunological, Infections immunology, Killer Cells, Natural immunology
- Abstract
Purpose of Review: As the molecular networks that connect innate and adaptive immunity are untangled, the prominence of natural killer (NK) cells in host defense continues to emerge. Herein we highlight recent findings pertaining to NK cell development, trafficking, and interactions with other innate and adaptive immune cells in the context of predicting how NK cells may be involved in a wider range of clinical immunodeficiency., Recent Findings: NK cells contribute vital roles in innate and adaptive immunity, especially in collaboration with dendritic cells (DC). Fascinating new details have been reported about cell surface integrins and receptors that regulate NK functions, as well as the cytokine/chemokine networks that provide for NK-DC interactions. Moreover, NK cells appear to play an important role in the attenuation or resolution of an immune response through either action against CD8 T cells or indirect control of certain DC. These findings shed important insights as to how NK cells and DC cooperate to control primary infections and shape the subsequent adaptive immune responses., Summary: Natural killer cells are heterogeneous lymphocytes that provide an essential function in host defense. NK cells respond early to microbial assault and interact with other cells of the innate immune system, but they recognize and intercept pathogenic infections through highly specific mechanisms that are similar to T cells. Thus, NK cells are positioned as a cellular bridge between innate and adaptive immunity. It is imperative, then, to include a careful assessment of NK cell populations and functions in most cases of suspected immunodeficiency.
- Published
- 2003
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33. Recognition of a virus-encoded ligand by a natural killer cell activation receptor.
- Author
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Smith HR, Heusel JW, Mehta IK, Kim S, Dorner BG, Naidenko OV, Iizuka K, Furukawa H, Beckman DL, Pingel JT, Scalzo AA, Fremont DH, and Yokoyama WM
- Subjects
- Animals, Ligands, Mice, Open Reading Frames, Killer Cells, Natural immunology, Muromegalovirus immunology, Receptors, Cell Surface immunology
- Abstract
Natural killer (NK) cells express inhibitory and activation receptors that recognize MHC class I-like molecules on target cells. These receptors may be involved in the critical role of NK cells in controlling initial phases of certain viral infections. Indeed, the Ly49H NK cell activation receptor confers in vivo genetic resistance to murine cytomegalovirus (MCMV) infections, but its ligand was previously unknown. Herein, we use heterologous reporter cells to demonstrate that Ly49H recognizes MCMV-infected cells and a ligand encoded by MCMV itself. Exploiting a bioinformatics approach to the MCMV genome, we find at least 11 ORFs for molecules with previously unrecognized features of predicted MHC-like folds and limited MHC sequence homology. We identify one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-mediated cytotoxicity, and cytokine and chemokine production by freshly isolated NK cells. We hypothesize that the other ORFs with predicted MHC-like folds may be involved in immune evasion or interactions with other NK cell receptors.
- Published
- 2002
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34. Vital involvement of a natural killer cell activation receptor in resistance to viral infection.
- Author
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Brown MG, Dokun AO, Heusel JW, Smith HR, Beckman DL, Blattenberger EA, Dubbelde CE, Stone LR, Scalzo AA, and Yokoyama WM
- Subjects
- Animals, Antibodies, Monoclonal immunology, Crosses, Genetic, Cytotoxicity, Immunologic, Female, Haplotypes, Histocompatibility Antigens Class I immunology, Humans, Lectins, C-Type, Ligands, Male, Membrane Glycoproteins genetics, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Phenotype, Receptors, NK Cell Lectin-Like, Tumor Cells, Cultured, Antigens, Ly, Herpesviridae Infections immunology, Immunity, Innate, Killer Cells, Natural immunology, Lymphocyte Activation, Membrane Glycoproteins immunology, Muromegalovirus immunology, Receptors, Immunologic immunology
- Abstract
Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.
- Published
- 2001
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35. Nonstochastic coexpression of activation receptors on murine natural killer cells.
- Author
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Smith HR, Chuang HH, Wang LL, Salcedo M, Heusel JW, and Yokoyama WM
- Subjects
- Animals, Antibodies, Monoclonal pharmacology, Antibody Specificity, Base Sequence, Cell Line, Cross-Linking Reagents pharmacology, Cytotoxicity, Immunologic, DNA Primers genetics, Gene Expression, Humans, Killer Cells, Natural classification, Lectins, C-Type, Membrane Glycoproteins genetics, Membrane Glycoproteins immunology, Mice, Mice, Inbred BALB C, Models, Biological, Receptors, NK Cell Lectin-Like, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Antigens, Ly, Killer Cells, Natural immunology, Receptors, Immunologic genetics
- Abstract
Murine natural killer cells (NK) express lectin-like activation and inhibitory receptors, including the CD94/NKG2 family of receptors that bind Qa-1, and the Ly-49 family that recognizes major histocompatibility complex class I molecules. Here, we demonstrate that cross-linking of NK cells with a new specific anti-Ly-49H mAb induced NK cell cytotoxicity and cytokine production. Ly-49H is expressed on a subset of NK cells and can be coexpressed with Ly-49 inhibitory receptors. However, unlike Ly-49 inhibitory receptors, Ly-49H is not detectable on naive splenic CD3(+) T cells, indicating that Ly-49H may be an NK cell-specific activation receptor. In further contrast to the stochastically expressed Ly-49 inhibitory receptors, Ly-49H is preferentially expressed with the Ly-49D activation receptor, and expression of both Ly-49H and Ly-49D is augmented on NK cells that lack receptors for Qa-1 tetramers. On developing splenic NK1.1(+) cells, Ly-49D and Ly-49H are expressed later than the inhibitory receptors. These results directly demonstrate that Ly-49H activates primary NK cells, and suggest that expression of Ly-49 activation receptors by NK cells may be specifically regulated on NK cell subsets. The simultaneous expression of multiple activation receptors by individual NK cells contrasts with that of T cell antigen receptors and is relevant to the role of NK cells in innate immunity.
- Published
- 2000
- Full Text
- View/download PDF
36. Development of intra-natural killer complex (NKC) recombinant and congenic mouse strains for mapping and functional analysis of NK cell regulatory loci.
- Author
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Scalzo AA, Brown MG, Chu DT, Heusel JW, Yokoyama WM, and Forbes CA
- Subjects
- Animals, Cytomegalovirus immunology, Cytomegalovirus Infections genetics, Cytomegalovirus Infections immunology, Cytomegalovirus Infections virology, Female, Genotype, Immunity, Innate genetics, Killer Cells, Natural metabolism, Male, Mice, Mice, Congenic, Mice, Inbred BALB C, Phenotype, Spleen virology, Viral Load, Chromosome Mapping, Crosses, Genetic, Genes, Killer Cells, Natural immunology, Recombination, Genetic
- Published
- 1999
- Full Text
- View/download PDF
37. Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells.
- Author
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Ho EL, Heusel JW, Brown MG, Matsumoto K, Scalzo AA, and Yokoyama WM
- Subjects
- Amino Acid Sequence, Animals, Antigens, CD immunology, Humans, Membrane Glycoproteins immunology, Mice, Molecular Sequence Data, Multigene Family, NK Cell Lectin-Like Receptor Subfamily D, Receptors, Immunologic immunology, Sequence Alignment, Antigens, CD genetics, Gene Expression Regulation immunology, Killer Cells, Lymphokine-Activated immunology, Killer Cells, Natural immunology, Lectins, C-Type, Membrane Glycoproteins genetics, Receptors, Immunologic genetics
- Abstract
Natural killer (NK) cells express C-type lectin-like receptors, encoded in the NK gene complex, that interact with major histocompatibility complex class I and either inhibit or activate functional activity. Human NK cells express heterodimers consisting of CD94 and NKG2 family molecules, whereas murine NK cells express homodimers belonging to the Ly-49 family. The corresponding orthologues for other species, however, have not been described. In this report, we used probes derived from the expressed sequence tag database to clone C57BL/6-derived cDNAs homologous to human NKG2-D and CD94. Among normal tissues, murine NKG2-D and CD94 transcripts are highly expressed only in activated NK cells, including both Ly-49A+ and Ly-49A- subpopulations. Additionally, mNKG2-D is expressed in murine NK cell clones KY-1 and KY-2, whereas mCD94 expression is observed only in KY-1 cells but not KY-2. Last, we have finely mapped the physical location of the Cd94 (centromeric) and Nkg2d (telomeric) genes between Cd69 and the Ly49 cluster in the NK complex. Thus, these data indicate the expanding complexity of the NK complex and the corresponding repertoire of C-type lectin-like receptors on murine NK cells.
- Published
- 1998
- Full Text
- View/download PDF
38. Long-range disruption of gene expression by a selectable marker cassette.
- Author
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Pham CT, MacIvor DM, Hug BA, Heusel JW, and Ley TJ
- Subjects
- Animals, Cathepsin G, Cathepsins genetics, Gene Library, Granzymes, Kanamycin Kinase, Mice, Mice, Knockout, Molecular Sequence Data, Mutagenesis, Insertional, Phosphoglycerate Kinase biosynthesis, Phosphoglycerate Kinase genetics, Phosphotransferases (Alcohol Group Acceptor) biosynthesis, Polymerase Chain Reaction, Promoter Regions, Genetic, Regulatory Sequences, Nucleic Acid, DNA Transposable Elements, Gene Expression, Genetic Markers, Globins genetics, Multigene Family, Serine Endopeptidases genetics
- Abstract
Recent studies have suggested that the retention of selectable marker cassettes (like PGK-Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in "knockout" mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the beta-like globin gene cluster. The insertion of PGK-Neo into the granzyme B gene, the most 5' gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK-Neo cassette into the beta-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3' to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these-long distance effects are unknown, the expression of PGK-Neo can be "captured" by the regulatory domain into which it is inserted. These results suggest that the PGK-Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.
- Published
- 1996
- Full Text
- View/download PDF
39. Granzyme B plays a critical role in cytotoxic lymphocyte-induced apoptosis.
- Author
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Shresta S, Heusel JW, Macivor DM, Wesselschmidt RL, Russell JH, and Ley TJ
- Subjects
- Animals, Granzymes, Killer Cells, Natural immunology, Mice, T-Lymphocytes, Cytotoxic immunology, Apoptosis immunology, Cytotoxicity, Immunologic, Killer Cells, Natural enzymology, Serine Endopeptidases immunology, T-Lymphocytes, Cytotoxic enzymology
- Published
- 1995
- Full Text
- View/download PDF
40. Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells.
- Author
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Shresta S, MacIvor DM, Heusel JW, Russell JH, and Ley TJ
- Subjects
- Amino Acid Sequence, Animals, Cells, Cultured, Cytotoxicity, Immunologic, Granzymes, Mice, Molecular Sequence Data, Apoptosis immunology, Killer Cells, Lymphokine-Activated immunology, Killer Cells, Natural immunology, Serine Endopeptidases metabolism
- Abstract
Granzyme (Gzm) B-deficient mice obtained by gene targeting were used to assess the role of Gzm B in the mechanisms used by natural killer (NK) and lymphokine-activated killer (LAK) cells to destroy target cells. Gzm B-/- NK cells, LAK cells, and cytotoxic T lymphocytes (CTL) all are defective in their ability to rapidly induce DNA fragmentation/apoptosis in susceptible target cells. This defect can be partially corrected with long incubation times of effector and target cells. Moreover, Gzm B-/- NK cells (but not CTL or LAK cells) exhibit a defect in 51Cr release from susceptible target cells. This 51Cr release defect in Gzm B-deficient NK cells is also not overcome by prolonged incubation times or high effector-to-target cell ratios. We conclude that Gzm B plays a critical and nonredundant role in the rapid induction of DNA fragmentation/apoptosis by NK cells, LAK cells, and CTL. Gzm B may have an additional role in NK cells (but not in CTL or LAK cells) for mediating 51Cr release.
- Published
- 1995
- Full Text
- View/download PDF
41. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells.
- Author
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Heusel JW, Wesselschmidt RL, Shresta S, Russell JH, and Ley TJ
- Subjects
- Animals, Embryo, Mammalian cytology, Granzymes, Hematopoiesis physiology, Killer Cells, Natural enzymology, Lymphocyte Activation physiology, Lymphocyte Culture Test, Mixed, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Multigene Family, Mutation, Serine Endopeptidases genetics, Stem Cells, T-Lymphocytes, Cytotoxic enzymology, Apoptosis physiology, DNA metabolism, Killer Cells, Natural physiology, Serine Endopeptidases physiology, T-Lymphocytes, Cytotoxic physiology
- Abstract
We have generated H-2b mice with a homozygous null mutation in the granzyme (gzm) B gene. Gzm B is a neutral serine protease with Aspase activity that is found only in the granules of activated cytolytic T cells, natural killer cells, and lymphokine-activated killer cells. Gzm B-/- mice develop normally and have normal hematopoiesis and lymphopoiesis. In vitro, cytotoxic T lymphocytes (CTL) derived from gzm B-/- animals are able to induce 51Cr release from allotarget cells, but with reduced efficiency. However, gzm B-/- CTL have a profound defect in their ability to induce rapid DNA fragmentation and apoptosis in allogeneic target cells. This defect is kinetic since DNA fragmentation is partially compensated and 51Cr release is completely rescued with long incubation times. We conclude that gzm B serves a critical and nonredundant role for the rapid induction of target cell DNA fragmentation and apoptosis by alloreactive cytotoxic T lymphocytes.
- Published
- 1994
- Full Text
- View/download PDF
42. Structure and expression of a cluster of human hematopoietic serine protease genes found on chromosome 14q11.2.
- Author
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Heusel JW, Hanson RD, Silverman GA, and Ley TJ
- Subjects
- Blotting, Southern, DNA genetics, Gene Expression Regulation, Genetic Linkage, Humans, Molecular Sequence Data, Restriction Mapping, Sequence Homology, Nucleic Acid, Chromosomes, Human, Pair 14, Lymphocytes enzymology, Multigene Family, Serine Endopeptidases genetics
- Abstract
We previously identified a cluster of hematopoietic serine protease genes on chromosome 14 at band q11.2. This cluster contains the cathepsin G gene and the two related cathepsin G-like genes CGL-1 and CGL-2. The CGL-1 gene is identical with the cytotoxic T cell serine protease CSP-B (also called SECT, and in mice, CCP1, granzyme B, or CTLA-1). In this report, we determined that CGL-2 is identical with a recently described gene called h-CCPX. The coding sequences of CG, CGL-1, and CGL-2 are 65-75% identical at the DNA level. The intervening sequences are much less conserved, except for introns 3 of the CGL-1 and CGL-2 genes, which are 93% identical. Each of the genes has the same overall organization, with 5 exons and 4 introns, very short 5' untranslated regions, and identical splice phases for all of the introns. Cathepsin G is expressed at high levels in promyelocytes/promonocytes, and CGL-1/CSP-B is expressed at high levels in activated cytolytic T cells, lymphokine-activated killer (LAK), and natural killer (NK) cells. CGL-2/h-CCPX is expressed at much lower levels in activated peripheral blood lymphocytes, LAK and NK cells. To begin to define the regulatory elements that target expression of each of these genes to their specific lineages at specific times, the 5' flanking region of each gene was sequenced. The 5' flanking regions are minimally related and have few conserved consensus elements. Further experiments will be required to determine the critical cis-acting regulatory sequences required for tissue- and development-specific expression of each of these genes.
- Published
- 1991
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