Aaqib Zaffar Banday1, Anit Kaur2, Tadayuki Akagi3, Dharmagat Bhattarai2, Masahiro Muraoka4,5, Diksha Dev6, Jhumki Das2, Man Updesh Singh Sachdeva6, Indrani Karmakar6, Kanika Arora2, Gurjit Kaur2, Vignesh Pandiarajan2, Ankur Kumar Jindal2, Taizo Wada4, H Phillip Koeffler5,7, Deepti Suri2, Jasmina Ahluwalia6, Hirokazu Kanegane8, Prateek Bhatia9, Amit Rawat10, Surjit Singh2. 1. Allergy Immunology Unit, Department of Pediatrics, Advanced Pediatrics Center (APC), Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, 160012, India. maaqibzb@gmail.com. 2. Allergy Immunology Unit, Department of Pediatrics, Advanced Pediatrics Center (APC), Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, 160012, India. 3. Department of Life, Environment, and Applied Chemistry, Faculty of Engineering, Fukuoka Institute of Technology, Fukuoka, Japan. 4. Department of Pediatrics, School of Medicine Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan. 5. Cedars-Sinai Medical Center, Division of Hematology/Oncology, UCLA School of Medicine, Los Angeles, CA, USA. 6. Department of Hematology, Research Block-A, PGIMER, Chandigarh, India. 7. Cancer Science Institute, National University of Singapore, Singapore, Singapore. 8. Department of Child Health and Development, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan. 9. Department of Pediatrics, Hematology Unit, APC, PGIMER, Chandigarh, India. 10. Allergy Immunology Unit, Department of Pediatrics, Advanced Pediatrics Center (APC), Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, 160012, India. rawatamit@yahoo.com.
Abstract
PURPOSE: Specific granule deficiency (SGD) is a rare inborn error of immunity resulting from loss-of-function variants in CEBPE gene (encoding for transcription factor C/EBPε). Although this genetic etiology has been known for over two decades, only a few patients with CEBPE variant-proven SGD (type I) have been reported. Herein, we describe two siblings with a novel homozygous CEBPE deletion who were noted to have profound neutropenia on initial evaluation. We aimed to evaluate the immunohematological consequences of this novel variant, including profound neutropenia. METHODS: Light scatter characteristics of granulocytes were examined on various automated hematology analyzers. Phagocyte immunophenotype, reactive oxygen species generation, and Toll-like receptor (TLR) signaling were assessed using flow cytometry. Relative expression of genes encoding various granule proteins was studied using RT-PCR. Western blot analysis and luciferase reporter assay were performed to explore variant C/EBPε expression and function. RESULTS: Severe infections occurred in both siblings. Analysis of granulocyte light scatter plots revealed automated hematology analyzers can provide anomalously low neutrophil counts due to abnormal neutrophil morphology. Neutrophils displayed absence/marked reduction of CD15/CD16 expression and overexpression (in a subset) of CD14/CD64. Three distinct populations of phagocytes with different oxidase activities were observed. Impaired shedding of CD62-ligand was noted on stimulation with TLR-4, TLR-2/6, and TLR-7/8 agonists. We demonstrated the variant C/EBPε to be functionally deficient. CONCLUSION: Homozygous c.655_665del variant in CEBPE causes SGD. Anomalous automated neutrophil counts may be reported in patients with SGD type I. Aberrant TLR signaling might be an additional pathogenetic mechanism underlying immunodeficiency in SGD type I.
PURPOSE: Specific granule deficiency (SGD) is a rare inborn error of immunity resulting from loss-of-function variants in CEBPE gene (encoding for transcription factor C/EBPε). Although this genetic etiology has been known for over two decades, only a few patients with CEBPE variant-proven SGD (type I) have been reported. Herein, we describe two siblings with a novel homozygous CEBPE deletion who were noted to have profound neutropenia on initial evaluation. We aimed to evaluate the immunohematological consequences of this novel variant, including profound neutropenia. METHODS: Light scatter characteristics of granulocytes were examined on various automated hematology analyzers. Phagocyte immunophenotype, reactive oxygen species generation, and Toll-like receptor (TLR) signaling were assessed using flow cytometry. Relative expression of genes encoding various granule proteins was studied using RT-PCR. Western blot analysis and luciferase reporter assay were performed to explore variant C/EBPε expression and function. RESULTS: Severe infections occurred in both siblings. Analysis of granulocyte light scatter plots revealed automated hematology analyzers can provide anomalously low neutrophil counts due to abnormal neutrophil morphology. Neutrophils displayed absence/marked reduction of CD15/CD16 expression and overexpression (in a subset) of CD14/CD64. Three distinct populations of phagocytes with different oxidase activities were observed. Impaired shedding of CD62-ligand was noted on stimulation with TLR-4, TLR-2/6, and TLR-7/8 agonists. We demonstrated the variant C/EBPε to be functionally deficient. CONCLUSION: Homozygous c.655_665del variant in CEBPE causes SGD. Anomalous automated neutrophil counts may be reported in patients with SGD type I. Aberrant TLR signaling might be an additional pathogenetic mechanism underlying immunodeficiency in SGD type I.
Authors: Maria T Larsen; Mattias Häger; Andreas Glenthøj; Fazila Asmar; Stine N Clemmensen; Helena Mora-Jensen; Niels Borregaard; Jack B Cowland Journal: Blood Date: 2014-01-07 Impact factor: 22.113