{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,2]],"date-time":"2026-11-02T17:59:32Z","timestamp":1793642372092,"version":"4.5.1"},"reference-count":30,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T00:00:00Z","timestamp":1755475200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>Chronic granulomatous disease is caused by mutations in any of the 6 components of the phagocytic NADPH oxidase complex including gp91<jats:sup>phox<\/jats:sup>, p47<jats:sup>phox<\/jats:sup>, p22<jats:sup>phox<\/jats:sup>, p40<jats:sup>phox<\/jats:sup>, p67<jats:sup>phox<\/jats:sup>, or EROS. Functional assays include reactive oxygen species (ROS) production, flow cytometry, and immunoblotting for NADPH proteins. The advent of high-throughput sequencing allows genetic diagnosis for all components except <jats:italic>NCF1<\/jats:italic> (p47<jats:sup>phox<\/jats:sup>) due to two, nearly identical, pseudogenes (<jats:italic>NCF1B<\/jats:italic>, <jats:italic>NCF1C<\/jats:italic>). The majority of NCF1-CGD patients carry a 2-base deletion caused by crossover between <jats:italic>NCF1<\/jats:italic> and <jats:italic>NCF1B<\/jats:italic> or <jats:italic>NCF1C<\/jats:italic>. Currently, NCF1 deficiency is diagnosed functionally: a characteristic DHR with low levels of residual ROS, loss of p47<jats:sup>phox<\/jats:sup> on immunoblot, or digital droplet PCR or Gene-scan to enumerate intact (GTGT) or deleted (\u0394GT). While this provides patients a clinical CGD diagnosis, for the 20% of NCF1-CGD patients with a non-\u0394GT mutation a definitive genetic diagnosis is still lacking.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We developed a bioinformatic method using existing short or long-read sequencing data from 48 NCF1-CGD patients or carriers.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>We identified both \u0394GT and non-\u0394GT <jats:italic>NCF1<\/jats:italic> gene mutations. Additionally, we confirm that the presence of \u0394GT in <jats:italic>NCF1<\/jats:italic> is due to pseudogene copy into the <jats:italic>NCF1<\/jats:italic> locus. We compare <jats:italic>NCF1<\/jats:italic> sequence from NCF1-CGD patients to cohorts of non-NCF1-CGD and healthy controls (1000Genomes), demonstrating pseudogene replacement of <jats:italic>NCF1<\/jats:italic> in NCF1-CGD as well as the reciprocal replacement of <jats:italic>NCF1B<\/jats:italic> or <jats:italic>NCF1C<\/jats:italic> by <jats:italic>NCF1<\/jats:italic> in some healthy controls.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>With this method, reanalysis of existing sequence data may provide genetic diagnosis to NCF1-CGD patients. This technique may be modified for other diagnostically relevant pseudogenes.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2025.1640496","type":"journal-article","created":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T05:29:29Z","timestamp":1755494969000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["Reliable genetic diagnosis of NCF1 (p47phox)-deficient chronic granulomatous disease using high-throughput sequencing"],"prefix":"10.3389","volume":"16","author":[{"given":"Amy P.","family":"Hsu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric","family":"Karlins","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Justin","family":"Lack","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"T. Joseph","family":"Pepper","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karen","family":"Lau","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kimberly R.","family":"Marshall-Batty","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Debra","family":"Long Priel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joie","family":"Davis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danielle L.","family":"Fink","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christa S.","family":"Zerbe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John I.","family":"Gallin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Harry L.","family":"Malech","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven M.","family":"Holland","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Douglas B.","family":"Kuhns","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,8,18]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1007097","article-title":"Residual NADPH oxidase and survival in chronic granulomatous disease","volume":"363","author":"Kuhns","year":"2010","journal-title":"N Engl J Med"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1182\/bloodadvances.2018023184","article-title":"NCF1 (p47phox)-deficient chronic granulomatous disease: comprehensive genetic and flow cytometric analysis","volume":"3","author":"Kuhns","year":"2019","journal-title":"Blood Adv"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1007\/s10875-008-9243-y","article-title":"First report of clinical, functional, and molecular investigation of chronic granulomatous disease in nine Jordanian families","volume":"29","author":"Bakri","year":"2009","journal-title":"J Clin Immunol"},{"key":"B4","doi-asserted-by":"publisher","first-page":"792","DOI":"10.1007\/s10875-011-9567-x","article-title":"Inheritance pattern and clinical aspects of 93 Iranian patients with chronic granulomatous disease","volume":"31","author":"Fattahi","year":"2011","journal-title":"J Clin Immunol"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1016\/S0301-472X(01)00731-7","article-title":"Gene-scan method for the recognition of carriers and patients with p47(phox)-deficient autosomal recessive chronic granulomatous disease","volume":"29","author":"Dekker","year":"2001","journal-title":"Exp Hematol"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1002\/humu.20413","article-title":"Chronic granulomatous disease caused by mutations other than the common GT deletion in NCF1, the gene encoding the p47phox component of the phagocyte NADPH oxidase","volume":"27","author":"Roos","year":"2006","journal-title":"Hum Mutat"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.88.7.2753","article-title":"Autosomal recessive chronic granulomatous disease caused by deletion at a dinucleotide repeat","volume":"88","author":"Casimir","year":"1991","journal-title":"Proc Natl Acad Sci U S A."},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1182\/blood.V95.6.2150","article-title":"Recombination events between the p47-phox gene and its highly homologous pseudogenes are the main cause of autosomal recessive chronic granulomatous disease","volume":"95","author":"Roesler","year":"2000","journal-title":"Blood."},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtm.2019.02.001","article-title":"Novel Diagnostic tool for p47phox-deficient chronic granulomatous disease patient and carrier detection","volume":"13","author":"Wrona","year":"2019","journal-title":"Mol Ther Methods Clin Dev"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btq033","article-title":"BEDTools: a flexible suite of utilities for comparing genomic features","volume":"26","author":"Quinlan","year":"2010","journal-title":"Bioinformatics."},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1172\/JCI124011","article-title":"Rescue of recurrent deep intronic mutation underlying cell type-dependent quantitative NEMO deficiency","volume":"129","author":"Boisson","year":"2019","journal-title":"J Clin Invest."},{"key":"B12","doi-asserted-by":"publisher","first-page":"201178","DOI":"10.1101\/201178","article-title":"Scaling accurate genetic variant discover to tens of thousands of samples","author":"Poplin","year":"2017","journal-title":"BioRxiv"},{"key":"B13","article-title":"Validation of bioinformatics pipeline to detect NEMO-deleted exon 5 autoinflammatory syndrome (NEMO-NDAS) and preliminary clinical and immunologic characterization [abstract","volume":"73","author":"Almeida de Jesus","year":"2021","journal-title":"Arthritis Rheumatol"},{"key":"B14","doi-asserted-by":"publisher","first-page":"102596","DOI":"10.1016\/j.bcmd.2021.102596","article-title":"Hematologically important mutations: The autosomal forms of chronic granulomatous disease (third update)","volume":"92","author":"Roos","year":"2021","journal-title":"Blood Cells Mol Dis"},{"key":"B15","doi-asserted-by":"publisher","first-page":"92","DOI":"10.1038\/s41586-023-06045-0","article-title":"A genomic mutational constraint mamp using variation in 76,156 human genomes","volume":"625","author":"Chen","year":"7993","journal-title":"Nature."},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkad989","article-title":"CADD v1.7: Using protein language models, regulator CNNs and other nucleotide-level scores to improve genome-wide variant predictions","volume":"52","author":"Schubach","year":"2024","journal-title":"Nucleic Acids Res"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1093\/bib\/bbs017","article-title":"Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration","volume":"14","author":"Thorvaldsdottir","year":"2013","journal-title":"Brief Bioinform"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1001\/jama.233.12.1295","article-title":"Chronic granulomatous disease. Diagnosis in a 27-year-old man with Mycobacterium fortuitum","volume":"233","author":"Chusid","year":"1975","journal-title":"JAMA."},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbadis.2013.05.001","article-title":"Different unequal cross-over events between NCF1 and its pseudogenes in autosomal p47(phox)-deficient chronic granulomatous disease","volume":"1832","author":"Hayrapetyan","year":"2013","journal-title":"Biochim Biophys Acta"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1182\/blood.V97.1.305","article-title":"Autosomal recessive chronic granulomatous disease caused by defects in NCF-1, the gene encoding the phagocyte p47-phox: mutations not arising in the NCF-1 pseudogenes","volume":"97","author":"Noack","year":"2001","journal-title":"Blood."},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.639226","article-title":"Second report of chronic granulomatous disease in Jordan: Clinical and genetic description of 31 patients from 21 different families, including families from Lybia and Iraq","volume":"12","author":"Bakri","year":"2021","journal-title":"Front Immunol"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1182\/bloodadvances.2016001214","article-title":"Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease","volume":"1","author":"Merling","year":"2016","journal-title":"Blood Adv"},{"key":"B23","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1186\/s12969-021-00536-y","article-title":"Homozygous variant p.Arg90His in NCF1 is associated with early-onset interferonopathy: a case report","volume":"19","author":"Schnappauf","year":"2021","journal-title":"Pediatr Rheumatol Online J"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1172\/JCI153619","article-title":"The NCF1 variant p.R90H aggravates autoimmunity by facilitating the activation of plasmacytoid dendritic cells","volume":"132","author":"Meng","year":"2002","journal-title":"J Clin Invest"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1086\/520985","article-title":"Fulminant mulch pneumonitis: an emergency presentation of chronic granulomatous disease","volume":"45","author":"Siddiqui","year":"2007","journal-title":"Clin Infect Dis"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.12659\/AJCR.902764","article-title":"Chronic granulomatous disease presenting as. Aspergillus fumigatus pneumonia in a previously healthy young woman","volume":"18","author":"Williams","year":"2017","journal-title":"Am J Case Rep"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2018.01258","article-title":"Chronic granulomatous disease first diagnosed in adulthood presenting with spinal cord infection","volume":"9","author":"Schwenkenbecher","year":"2018","journal-title":"Front Immunol"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1016\/j.jacig.2022.06.004","article-title":"Chronic granulomatous disease presenting at age 52 with fulminant mulch pneumonitis","volume":"1","author":"Marois","year":"2022","journal-title":"J Allergy Clin Immunol Glob"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1016\/S1081-1206(10)61212-4","article-title":"Newly diagnosed chronic granulomatous disease in a 53-year-old woman with Crohn disease","volume":"95","author":"Ramanuja","year":"2005","journal-title":"Ann Allergy Asthma Immunol"},{"key":"B30","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1186\/1710-1492-9-9","article-title":"Newly diagnosed chronic granulomatous disease in a 44 year old male presenting with recurrent groin cellulitis and colitis","volume":"9","author":"Chung","year":"2013","journal-title":"Allergy Asthma Clin Immunol"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1640496\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,9]],"date-time":"2025-09-09T10:36:28Z","timestamp":1757414188000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1640496\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,18]]},"references-count":30,"alternative-id":["10.3389\/fimmu.2025.1640496"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1640496","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,18]]},"article-number":"1640496"}}