Janus kinase inhibition for autoinflammation in patients with DNASE2 deficiency
Authors: Ying Hong, Melania Capitani, Claire Murphy, Sumeet Pandey, Athena Cavounidis, Haruo Takeshita, Sira Nanthapisal, Toshihiro Yasuda, Brigitte Bader-Meunier, Dara McCreary, Ebun Omoyinmi, Anupama Rao, Claire Booth, Kimberly Gilmour, Neil Sebire, Neil Shah, Nigel Klein, Alex N. Bullock, Despina Eleftheriou, Holm H. Uhlig, Paul Brogan
Journal: Journal of Allergy and Clinical Immunology (2019)

Defective elimination of nucleic acids can trigger autoinflammatory and autoimmune processes.1Lazear H.M. Schoggins J.W. Diamond M.S. Shared and distinct functions of type I and type III interferons.Immunity. 2019; 50: 907-923Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar Mice lacking the lysosomal endonuclease DNase II have shown intracellular DNA accumulation, leading to severe anemia and embryonic death.2Kawane K. Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver.Science. 2001; 292: 1546-1549Crossref PubMed Scopus (297) Google Scholar Human subjects carrying homozygous nonsynonymous loss-of-function mutations in DNASE2 complete gestation but are severely affected by congenital anemia and an inflammatory disorder characterized by splenomegaly, glomerulonephritis, liver fibrosis, circulating anti-DNA autoantibodies, and progressive arthritis.3Rodero M.P. Tesser A. Bartok E. Rice G.I. Della Mina E. Depp M. et al.Type I interferon-mediated autoinflammation due to DNase II deficiency.Nat Commun. 2017; 8: 2176Crossref PubMed Scopus (129) Google Scholar We describe a patient with a novel hypomorphic homozygous missense mutation in DNASE2 (c.A284G; p.Y95C) resulting in severe autoinflammation, failure to thrive, hemophagocytic lymphohistiocytosis (HLH), early-onset chronic intestinal inflammation, and suppression of hematopoiesis. Beyond the recent description of this disorder in 3 patients,3Rodero M.P. Tesser A. Bartok E. Rice G.I. Della Mina E. Depp M. et al.Type I interferon-mediated autoinflammation due to DNase II deficiency.Nat Commun. 2017; 8: 2176Crossref PubMed Scopus (129) Google Scholar we further characterize cytokine dysregulation and demonstrate successful treatment with the Janus kinase (JAK) 1/2 inhibitor baricitinib. The proband (V-2) is a 14-year-old Somalian girl from an endogamous kindred with a history of neonatal anemia (Fig 1, A). At 6 years of age, patient V-2 was referred for pyrexia, a history of pancytopenia, hepatosplenomegaly, failure to thrive (height and weight less than the 0.4th percentile), chronic diarrhea, and delayed motor skill development. Investigations (summarized in Table E1 in this article's Online Repository at www.jacionline.org) revealed severe pancytopenia requiring multiple transfusions of red cells and platelets. Intestinal histology from endoscopic biopsy specimens revealed multiple granuloma-like cells within mucosa-associated lymphoid tissue compatible with phagocytosed DNA fragments (pseudogranuloma). Magnetic resonance imaging of the brain revealed biparietal deep white matter parenchymal signal abnormalities (Fig 1, B). Bone marrow studies identified macrophage infiltration and associated hemophagocytosis (Fig 1, C). At 9 years of age, the patient experienced severe EBV infection, necessitating treatment with dexamethasone and rituximab. Typical HLH criteria were met (see Table E1). She responded partially to the HLH-2004 chemotherapy treatment protocol, which she received for 7 weeks,4Henter J.-I. Horne A. Aricó M. Egeler R.M. Filipovich A.H. Imashuku S. et al.HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis.Pediatr Blood Cancer. 2007; 48: 124-131Crossref PubMed Scopus (3219) Google Scholar with cessation of fever and improvement in diarrhea but ongoing pancytopenia and hepatosplenomegaly. For several years, transfusion-dependent anemia, chronic diarrhea, and severe growth retardation were observed. Homozygosity mapping and whole-exome sequencing revealed a rare homozygous variant in DNASE2 (c.284A>G; p.Y95C; see Fig E1, A, and Table E2 in this article's Online Repository at www.jacionline.org). The affected variant is highly conserved across species (Fig 1, D). We used structural data of the existing DNase II homodimer complexed with a double-stranded DNA crystal structure from Burkholderia thailandensis to model the human DNase II.5Varela-Ramirez A. Abendroth J. Mejia A.A. Phan I.Q. Lorimer D.D. Edwards T.E. et al.Structure of acid deoxyribonuclease.Nucleic Acids Res. 2017; 45: 6217-6227Crossref PubMed Scopus (3) Google Scholar Residue Y95 is closely packed with residue T114, which is central to the nuclease catalytic HxK motif (H113 and K115), suggesting that small perturbations at residue 95 might have a significant effect on catalytic activity. The 2 recently published mutations in DNASE2 (p.G116A and p.D121V)3Rodero M.P. Tesser A. Bartok E. Rice G.I. Della Mina E. Depp M. et al.Type I interferon-mediated autoinflammation due to DNase II deficiency.Nat Commun. 2017; 8: 2176Crossref PubMed Scopus (129) Google Scholar will likely disrupt the same catalytic domain (Fig 1, E). The p.Y95C variant completely abolishes DNase II activity in a single radial enzyme diffusion (see Fig E1, B) and confers loss of function of the protein rather than absence (see Fig E1, C and D). Furthermore, granulocyte lysates from patient V-2 confirmed a deficiency in degrading DNA (Fig 1, F). Macrophages show the greatest expression of DNASE2 and are most likely affected in DNase II–deficient patients (see Fig E2, A, in this article's Online Repository at www.jacionline.org). We observed increased lysosomal DNA accumulation in monocyte-derived macrophages from patient V-2 compared with control cells (see Fig E2, B). Similarly, clearance of exogenous apoptotic or bacterially derived DNA was reduced in macrophages from patient V-2 (see Fig E2, C and D). To investigate whether DNase II deficiency causes a selective DNA degradation defect or broadly affects lysosomal function, we tested antimicrobial activity in macrophages from patient V-2 (see Fig E2, E). Normal antimicrobial activity suggests that the defect in patients with DNASE2 deficiency is restricted to the inability to clear DNA. Previous studies emphasized that deficiency in DNASE2 in human subjects and mice results in induction of interferon- and non–interferon-mediated inflammation.2Kawane K. Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver.Science. 2001; 292: 1546-1549Crossref PubMed Scopus (297) Google Scholar,3Rodero M.P. Tesser A. Bartok E. Rice G.I. Della Mina E. Depp M. et al.Type I interferon-mediated autoinflammation due to DNase II deficiency.Nat Commun. 2017; 8: 2176Crossref PubMed Scopus (129) Google Scholar,6Pawaria S. Moody K. Busto P. Nündel K. Choi C.-H. Ghayur T. et al.Cutting edge: DNase II deficiency prevents activation of autoreactive B cells by double-stranded DNA endogenous ligands.J Immunol. 2015; 194: 1403-1407Crossref PubMed Scopus (41) Google Scholar,7Kawane K. Tanaka H. Kitahara Y. Shimaoka S. Nagata S. Cytokine-dependent but acquired immunity-independent arthritis caused by DNA escaped from degradation.Proc Natl Acad Sci U S A. 2010; 107: 19432-19437Crossref PubMed Scopus (84) Google Scholar Increased levels of TNF, monocyte chemoattractant protein 1, IL-18, IFN-2α, IFN-β, and IFN-γ were detected in sera of patient V-2 (Fig 1, G). Increased levels of TNF, IL-1β, interferon-inducible protein 10, and IL-8 were observed in fibroblasts from patient V-2 (see Fig E3, A, in this article's Online Repository at www.jacionline.org). p.Y95C macrophages showed high levels of TNF and IL-18, modestly increased levels of IL-1β, and a clear induction of type I interferons (see Fig E3, B), which we also confirmed to be present in one of the previously described patients (see Fig E3, B).3Rodero M.P. Tesser A. Bartok E. Rice G.I. Della Mina E. Depp M. et al.Type I interferon-mediated autoinflammation due to DNase II deficiency.Nat Commun. 2017; 8: 2176Crossref PubMed Scopus (129) Google Scholar Type I interferons (IFN-α and IFN-β) bind to type I receptors (IFN-α/β receptor 1/2) and induce the activation of JAK1 and tyrosine kinase 2, leading to signal transducer and activator of transcription 1 and 2 phosphorylation and interferon-stimulated gene (ISG) expression.8Ivashkiv L.B. Donlin L.T. Regulation of type I interferon responses.Nat Rev Immunol. 2014; 14: 36-49Crossref PubMed Scopus (1766) Google Scholar Because the immunotype of patient V-2 was indicative of a strong ISG signature, we explored JAK inhibition as a therapeutic option. In vitro JAK1/JAK2 blockade with baricitinib reduced ISG expression in fibroblasts from control subjects and patient V-2 and control macrophages after IFN-α stimulation (see Fig E4, A and B, in this article's Online Repository at www.jacionline.org). In contrast, selective blockade of JAK2 using AG490 did not revert the ISG signature (see Fig E4, A). In healthy macrophages baricitinib treatment had stronger effects on ISG reduction compared with fibroblasts (see Fig E4, B). IFN-α–induced IL-18 levels were reduced after baricitinib or AG490 treatment (see Fig E4, C), suggesting a contribution of JAK2 signaling to IL-18 production in control macrophages. Because JAK inhibitors are effective in several interferonopathies9Sanchez G.A.M. Reinhardt A. Ramsey S. Wittkowski H. Hashkes P.J. Berkun Y. et al.JAK1/2 inhibition with baricitinib in the treatment of autoinflammatory interferonopathies.J Clin Invest. 2018; 128: 3041-3052Crossref PubMed Scopus (291) Google Scholar and baricitinib reverted the inflammatory signature in fibroblasts and macrophages in vitro, we treated our otherwise therapy-nonresponsive patient with baricitinib. We started with a 2-mg oral dose once daily, increasing weekly by 2 mg to achieve a final target dose of 6 mg (2 mg 3 times a day). Baricitinib was well tolerated with no adverse events and resulted in rapid and sustained clinical improvement (now for >6 months). In particular, the improved energy levels reported by the patient and the ability to attend school full-time were relevant patient-reported outcomes. Weight gain improved (Fig 2, A), and the patient underwent menarche 4 months after starting baricitinib. Blood parameters (Fig 2, B), gastrointestinal symptoms, and fecal calprotectin levels improved (Fig 2, B). Baricitinib decreased ISG expression in peripheral blood and cytokine levels compared with pretreatment levels (Fig 2, C-E). After 6 months of treatment, however, although the ISG signature indicated normalization of SIGLEC1 and ISG15, there was an upward trend of IFIT1, IFI44L, RSDA2, IL18, and IFI27, despite ongoing therapeutic response. Our data suggest that JAK1/2 inhibition is a rational and effective treatment for DNase II deficiency. In conclusion, we describe an additional patient with human DNase II deficiency who presented with HLH and early-onset intestinal inflammation, expanding the phenotypic spectrum of DNase II deficiency. We characterize the mechanisms underpinning the complex immunophenotype and show in a single patient that blocking JAK1/JAK2 with baricitinib can be used to curb pathologic interferon signaling caused by an inability to clear intracellular DNA. We thank the patients and their families, as well as healthy volunteers, for consenting to this research. We thank Yanick Crow, Jan Rehwinkel, Anne Bridgeman, Jonny Hertzog, and Julie Schulthess for critical input and research materials. This study was approved by the Bloomsbury Ethics Committee (no. 08H071382), the Oxford GI biobank (no. 09/H1204/30), and the Comité de Protection des Personnes (ID-RCB/EUDRACT 2014-A01017-40) in France. We obtained written informed consent from all family members and adolescent healthy control subjects with local ethics approval (REC 11/LO/0330). All 5 family members were genotyped with the Illumina cytoSNP12 chip (Illumina, San Diego, Calif). Two hundred nanograms of DNA was isothermally amplified overnight and then enzymatically fragmented. The fragmented DNA was incubated on a BeadChip overnight. These were imaged with the Illumina iScan System. Regions of homozygosity were identified by using the Illumina BeadStudio with the loss-of-heterozygosity detector plug-in (version 1.0.3). The minimum number of contiguous homozygous single nucleotide polymorphisms was set to 100. Whole-exome sequencing was completed by using the Illumina platform and sequenced on the Illumina HiSeq2000. Raw sequence data were aligned to the human reference genome by using the Burrows-Wheeler Aligner algorithm. Variant calling was performed with the Genome Analysis Toolkit, and variants were annotated with wANNOVAR. The DNASE2 variant was confirmed and familial segregation was ascertained by using PCR and Sanger sequencing with the following primers to amplify and sequence exon 3: forward, GCCTTCTCTTCCCTCTCTCC; reverse, GTCAGGGGTTACCTTGGAAAAAT. Sanger sequencing was performed with the AB3730 Analyzer with the BigDye v3.1 kit (Applied Biosystems, Foster City, Calif). Multiple DNASE2 sequences were aligned by using ClustalW2.E1Goujon M. McWilliam H. Li W. Valentin F. Squizzato S. Paern J. et al.A new bioinformatics analysis tools framework at EMBL-EBI.Nucleic Acids Res. 2010; 38(Web Server): W695-W699Crossref Scopus (1303) Google Scholar Data obtained from the sequence alignment are based on the following National Center for Biotechnology Information accession numbers: Homo sapiens, AAH10419.3; Rattus norvegicus, NP_612548.1; Pan troglodytes, XP_001170388.1; Canis lupus, XP_533902.3; Mus musculus, NP_034192.1; Drosophila melanogaster, NP_650672.1; Caenorhabditis elegans, NP_491414; Burkholderia thailandensis, WP_009895252.1; and Burkholderia pseudomallei, YP_111989.1. Structural models and figures were prepared by using the ICM software package (MolSoft, San Diego, Calif). A homology model for human DNase II was prepared by using the existing structure from B thailandensis (PDB 5UNB) as the model template. Double-stranded DNA (PDB 2BNA) was positioned manually at the dimer interface to indicate the potential DNA-binding surface, as previously highlighted.E2Varela-Ramirez A. Abendroth J. Mejia A.A. Phan I.Q. Lorimer D.D. Edwards T.E. et al.Structure of acid deoxyribonuclease.Nucleic Acids Res. 2017; 45: 6217-6227Crossref PubMed Scopus (10) Google Scholar All coordinate files were obtained from the Protein Data Bank (http://www.rcsb.org). PBMCs were isolated from 15 mL of blood from the patient and control subjects by using density gradient centrifugation (Lymphoprep; STEMCELL Technologies, Vancouver, British Columbia, Canada) and cultured in RPMI 1640 with glutamine (Sigma-Aldrich, St Louis, Mo) supplemented with 10% FCS. PBMCs (4 × 106) were plated, and after 2 hours, adherent monocytes were washed and cultured in the presence of 100 ng/mL macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ) for 5 days to differentiate them to macrophages. PBMCs were sorted with fluorescence-activated cell sorting from healthy donor blood by using canonical cell population surface markers: dendritic cell CD1c+ (clone B-Ly6; BD Biosciences, Franklin Lake, NJ), B-cell CD19+ (clone HIB19; BioLegend, San Diego, Calif) and CD8+ (clone RPA-T8; BD Biosciences), T-cell CD4+ (clone RM4-5; BioLegend), T memory cell CD45RO (clone UCHL1, BioLegend) and CCR7 (clone G043H7, BioLegend), T-cell CD8+ (clone RPA-T8; BD Biosciences), DR+11C- (clone B-Ly6; BD Biosciences), natural killer cell CD16+ (clone 3G8; BioLegend), myeloid dendritic cell CD141+ (clone M80; BioLegend), and monocyte CD14+ (clone M5E2; BioLegend). Granulocytes were purified from the precipitate after the blood gradient by removing red blood cells with ACK Lysing Buffer (Invitrogen, Carlsbad, Calif). Granulocytes (3.5 × 106) from the patient and control subjects were lysed in 40 μL of lysis buffer (25 mmol/L Tris-HCl [pH 7.6], 150 mmol/L NaCl, 5 mmol/L ethylenediamine tetra-acetic acid, 1% NP-40 or 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) at pH 5.5 in the presence of proteinase and phosphate inhibitors. Lysates were spun down at 14,000 rpm for 15 minutes, and supernatants were collected. Each lysate was split in 2: 20 μL incubated with 2 μL of 1-Kbp DNA ladder solution (Bioline, London, United Kingdom) and 20 μL without. Samples were incubated for 90 minutes at 37ºC. Ten microliters of each sample was loaded on agarose gel (1.5%) and run under an electric field at 110 V in the presence of TAE 1 × buffer for 1 hour. Gel pictures were acquired on the UVP BioImaging System (Analytik Jena US LLC). Gel densitometric analysis was done with ImageJ software (National Institutes of Health, Bethesda, Md). The human DNASE2 cDNA sequence was cloned into the expression vector pcDNA3.1 (+; Invitrogen)E3Takeshita H. Yasuda T. Iida R. Nakajima T. Hosomi O. Nakashima Y. et al.Identification of the three non-identical subunits constituting human deoxyribonuclease II.FEBS Lett. 1998; 440: 239-242Crossref PubMed Scopus (22) Google Scholar and used as a wild-type construct. The variant construct corresponding to p.Y95C in DNASE2 was inserted into the wild-type template by using the KOD-Plus Mutagenesis kit (Toyobo, Osaka, Japan). Nucleotide sequences of the construct were confirmed by using DNA sequence analysis. Purification of each construct used for transfection was performed with the Plasmid Midi Kit (Qiagen, Hilden, Germany). DNase II activity in the transfected COS-7 cells was assayed by using the single radial enzyme diffusion method with an LAS-3000 imaging analyzer (Fuji Film, Tokyo, Japan), according to our previous reports.E4Ueki M. Takeshita H. Fujihara J. Kimura-Kataoka K. Iida R. Yuasa I. et al.Genetic and expression analysis of all 7 non-synonymous single nucleotide polymorphisms in the human deoxyribonuclease II gene, with potential relevance to autoimmunity.Clin Chim Acta. 2010; 411: 92-98Crossref PubMed Scopus (12) Google Scholar The activity of wild-type DNase II was defined as 1.0, and that of the amino acid–substituted DNase II was expressed relative to the wild-type. Macrophages (2 × 105) from control subjects and the patient were seeded on a Cyto Chamber and left to adhere for 2 hours. Subsequently, they were stained with 4′-6-diamidino-2-phenylindole dihydrochloride (1 μg/mL; Sigma-Aldrich) for 20 minutes and washed twice with PBS. To evaluate the ability of macrophages to process DNA from engulfed apoptotic cells, they were cocultured for 4 hours with SYBR Green (1:10,000; Life Technologies, Grand Island, NY)–positive HEK 293 apoptotic cells at a 1:1 ratio. Apoptosis was induced by means of exposure under a UV lamp for 20 minutes after 4 hours of culture; the apoptotic status was confirmed by using fluorescence-activated cell sorting staining for Annexin V and 7-aminoactinomycin D. Moreover, to evaluate the ability of macrophages to process DNA from invasive Salmonella species, they were infected for 1 hour with SYTO 9 (50 nmol/L; Life Technologies)–positive bacteria at a multiplicity of infection of 1:10 and then left for 4 hours under gentamicin treatment. In all conditions, 2 hours before the end of incubation, macrophages were stained with LysoTracker Deep Red (Thermo Fisher, Waltham, Mass) 1:2000 solution. Cells were fixed with PBS–paraformaldehyde 4% solution for 10 minutes at 37°C and then washed. Slides were mounted with a glass coverslip by using VECTASHIELD solution (Vector Laboratories, Burlingame, Calif). Images were captured with a Nikon eclipse ME600 fluorescence microscope (Nikon, Tokyo, Japan) and analyzed with ImageJ software. Macrophages at day 5 of differentiation were detached with Versene solution (Gibco, Carlsbad, Calif) for 5 minutes and counted. Macrophages (2 × 104) were seeded per well in 96-well plates. Then they were infected with Salmonella typhimurium at a multiplicity of infection of 1:10 for 1 hour, followed by 2 hours of treatment with 100 μg/mL gentamicin (Sigma-Aldrich). Cells were then placed in lysates with 50 μL of Triton 1 × . Undiluted and diluted 1:10 and 1:100 cell lysates were plated in LB-agar plates that were incubated overnight at 37°C. Bacterial colonies were counted manually. Healthy donor macrophages (1 × 105 cells) and fibroblasts (1 × 105 cells) were either left untreated or treated with baricitinib (300 nmol/L; Cayman Chemical, Ann Arbor, Mich), AG490 (20 μmol/L; Calbiochem, Nottingham, United Kingdom), and filgotinib (200 nmol/L; Cayman Chemicals) for 1 hour in a 48-well plate. After treatment, both macrophages and fibroblasts were either left unstimulated or stimulated with IFN-α (10 U/mL; R&D Systems, Minneapolis, Minn) for 24 hours in the presence of the drug. Whole blood was collected into PAXgene tubes (BD Biosciences). We used a PreAnalytix RNA isolation kit for whole blood and the RNeasy Micro Kit (Qiagen) for RNA isolation from macrophages and fibroblasts. The RNA concentration was assessed with a Nanodrop (FLUOstar Omega; Labtech, Orternberg, Germany). Total RNA was retrotranscribed to cDNA by using the Reverse Transcription kit (Applied Biosystems). We performed quantitative PCR analysis using the iTaq Universal SYBR Green Supermix (172-5121; Bio-Rad Laboratories, Hercules, Calif) and the TaqMan Gene Expression Assay (FAM; Applied Biosystems). The relative abundance of each of 6 "interferon score" targets (IFL27, IFIT1, IFI44L, ISG15, and RSAD2) and other proinflammatory transcripts was normalized to the expression level of HPRT1 or RPLPO assessed by using Bio-Rad CFX Maestro software. Cytokine levels were measured in the sera of patients and control subjects and in supernatants of fibroblasts using a Meso Scale Discovery multiplex kit (Meso Scale Diagnostics, Rockville, Md), according to the manufacturer's instructions. Control sera was obtained from adolescent healthy control subjects with local ethics approval (REC reference 11/LO/0330; n = 24; 13 male subjects; median age, 15.7 years; range, 10.7-17.5 years). Undiluted macrophage supernatants were assayed for TNF, IL-1β, and IL-18 cytokines using the ProcartaPlex-Multiplex Immunoassay (Invitrogen). All statistical (ANOVA or the Mann-Whitney U test) graphs were produced with Prism software (version 8; GraphPad Software). All experiments were repeated at a minimum in triplicates, unless otherwise specified. P values of less than .05 were considered significant.Fig E2Macrophages with the homozygous DNASE2 p.Y95C variant have impaired DNA clearance ability. A, DNASE2 gene expression of sorted human cells from healthy control subjects reveals that macrophages show the greatest expression of DNASE2. **P < .01 and ***P < .0001. B, Confocal image (left) and quantification of DNA in lysosomes (right) of macrophages from the patient and control subjects stained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) and LysoTracker. There was increased lysosomal DNA accumulation in monocyte-derived macrophages from patient V-2 compared with control subjects. **P < .01. C and F, Confocal image (left) and quantification of exogenous DNA in lysosomes per cell (right) of macrophages from patients and control subjects stained with DAPI and then cocultured with SYBR Green–positive apoptotic HEK 293 cells (Fig 2, C) and prestained SYTO 9–Salmonella (multiplicity of infection = 20; Fig 2, D). Monocyte-derived macrophages from patient V-2 and 2 previously reported siblings with the homozygous p.G116A mutation showed reduced clearance of exogenous apoptotic or bacterially derived DNA. ***P < .001. E, Gentamicin protection assay of monocyte-derived macrophages from patient V-2 and control subjects showed normal antimicrobial activity. The graph represents pooled data of colony-forming unit quantification.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Proinflammatory cytokine profile in fibroblasts and macrophages derived from proband V-2 with the homozygous p.Y95C DNASE2 variant. A, Increased levels of cytokines (TNF, IL-1β, interferon-inducible protein 10 [IP-10], and IL-8) quantified by means of ELISA and upregulation of gene expression of the ISGs (IFIT1 and IFI44L) in fibroblasts from patient V-2 compared with control cells. B, Increased levels of cytokines (TNF, IL-18, and IL-1β) quantified by using Luminex and upregulation of gene expression of the ISGs (IFIT1 and IFI44L) in macrophages from patient V-2 compared with control cells. Gene expression of ISGs (IFIT1 and IFI44L) in macrophages from a patient with the homozygous DNASE2 p.G116A variant and healthy control macrophages was also performed and is presented for comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Effect of in vitro JAK1/JAK2 blockade on type I interferon signature and IL-18 expression. A, Quantitative RT-PCR of ISGs (IFIT1, IFI44L, RSDA2, SIGLEC1, and IFI27) from patient V-2 and control fibroblasts with and without IFN-α stimulation in the presence and absence of baricitinib (JAK1/JAK2 blockade), AG490 (selective JAK2 blockade), and filgotinib (selective JAK1 blockade) for 24 hours. B, Quantitative PCR of ISGs (IFIT1, IFI44L, RSDA2, SIGLEC1, and IFI27) from healthy donor macrophages stimulated with and without IFN-α in the presence and absence of baricitinib or AG490 for 24 hours. C, Quantitative PCR of IL18 and IL1B genes from healthy donor macrophages stimulated with and without IFN-α in the presence and absence of baricitinib and AG490 for 24 hours. **P < .01, ***P < .001, and ****P < .0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Patient V-2's laboratory investigationsLaboratory investigationsPatient V-2 (reference range)Autoantibodies persistent >3 moAbsent†Autoantibodies tested: anti-nuclear antibodies, anti-neutrophil cytoplasm antibodies, rheumatoid factor, anti-tissue transglutaminase antibodies, and anti-thyroid peroxidase antibodies.Hemoglobin∗Laboratory features contributing to the diagnosis of HLH. Other clinical features supporting the diagnosis of HLH included fever, hepatosplenomegaly, and hemophagocytosis demonstrated in bone marrow aspirate (see main text).50 g/L‡Lowest hemoglobin level of 147 measurements. (120-160 g/L)Platelet count∗Laboratory features contributing to the diagnosis of HLH. Other clinical features supporting the diagnosis of HLH included fever, hepatosplenomegaly, and hemophagocytosis demonstrated in bone marrow aspirate (see main text).21 × 109/L§Lowest platelet count of 147 measurements. (150-450 × 109/L)White blood cell count∗Laboratory features contributing to the diagnosis of HLH. Other clinical features supporting the diagnosis of HLH included fever, hepatosplenomegaly, and hemophagocytosis demonstrated in bone marrow aspirate (see main text).0.24 × 109/L‖Lowest white blood cell count of 147 measurements. (4.0-11 × 109/L)Triglycerides∗Laboratory features contributing to the diagnosis of HLH. Other clinical features supporting the diagnosis of HLH included fever, hepatosplenomegaly, and hemophagocytosis demonstrated in bone marrow aspirate (see main text).3.98 mmol/L (0.32-1.46 mmol/L)Fibrinogen2.9 g/L (1.7-4.0 g/L)Monocyte and lymphocyte subsetsLymphopenia¶Normal proportions of T, B, and natural killer cells but in low numbers.Normal αβ double-negative T cellsNormal γδ T cellsNaive T-cell subsetsVariable#Test repeated 4 times; normal proportion of naive cells on 3 occasions (54% to 64%) and numbers of reduced naive T cells for age on 1 occasion (35%).IgG19.9 g/L (3.1-13.8 g/L)IgA1.94 g/L (0.4-0.7 g/L)IgM2.29 g/L (0.5-2.2 g/L)IgD7 kU/L (2-100 kU/L)IgE5.1 kU/L (0-48 kU/L)MHC class I expressionNormalT-cell Vβ repertoireConsistent with polyclonal T cellsIL-2–inducible T-cell kinase ITK immunoblotNormalNitroblue tetrazolium testNormalVaccine responsesVariableIgG antibodies to tetanus normal (0.04 IU/mL; range, >0.1 IU/mL); IgG antibodies to pneumococcus (all <0.35 μg/mL; range, >0.35 μg/mL).Perforin expression (CD56 cells)NormalVery few cells available for analysis. Perforin was expressed, but quantification was not possible.Perforin gene sequencingNo damaging mutationsOne synonymous polymorphism in PRF1, c.C900T p.H330H, identified with an Exome Aggregation Consortium frequency of 63%.CD107a granule release (CD8 and NK cells)CD8 CD107a+: control, 5.0%; patient, 6.9%NK CD107a: control, 13.7%; patient, 23.1%Natural killer cytotoxicity test∗Laboratory features contributing to the diagnosis of HLH. Other clinical features supporting the diagnosis of HLH included fever, hepatosplenomegaly, and hemophagocytosis demonstrated in bone marrow aspirate (see main text).AbnormalTest only completed on 1 occasion, with subsequent efforts hampered by insufficient cells.Gene sequencing for known causes of HLH and immunodeficiencyTIGER (Targeted Immuno and Gastro Enrichment) panel covering 72 genes implicated in immunodeficiency and 53 genes implicated in diseases of interest to gastroenterology.No known disease-causing mutations identifiedT-cell receptor excision circles1902 (>10,000/million T cells)Low for age.Soluble CD25∗Laboratory features contributing to the diagnosis of HLH. Other clinical features supporting the diagnosis of HLH included fever, hepatosplenomegaly, and hemophagocytosis demonstrated in bone marrow aspirate (see main text).7624 pg/mLHighest of 2 repeats (range, 4301-4702 pg/mL). (<2500 pg/mL)∗Healthy children and adults have soluble CD25 levels of less than 2500 pg/mL. Children with inflammation have levels of 2500 pg/mL or greater. To date, genetically confirmed HLHs typically have had soluble CD25 levels of greater than 5000 pg/mL, but this decreases with treatment.Complement C31.49 g/L (0.75-1.65 g/L)Complement C40.45 g/L (0.14-0.54 g/L)Fecal calprotectin472 mg/kg†Greatest value of 2 measurements (range, 450-472 mg/kg). (<50 mg/kg)Ferritin∗Laboratory features contributing to the diagnosis of HLH. Other clinical features supporting the diagnosis of HLH included fever, hepatosplenomegaly, and hemophagocytosis demonstrated in bone marrow aspirate (see main text).7371 μg/L‡Greatest value of 43 measurements (7371 ng/mL). (12-81 μg/L)Liver enzymesAlanine aminotransferase, 219 U/L (10-25 U/L)Alkaline phosphatase, 96 U/L (150-380 U/L)PHA stimulationVariable§Test repeated 8 times, 2 times with normal results, 4 times absent, 1 time impaired, and 1 time unable to analyze.NK, Natural killer.∗ Laboratory features contributing to the diagnosis of HLH. Other clinical features supporting the diagnosis of HLH included fever, hepatosplenomegaly, and hemophagocytosis demonstrated in bone marrow aspirate (see main text).† Autoantibodies tested: anti-nuclear antibodies, anti-neutrophil cytoplasm antibodies, rheumatoid factor, anti-tissue transglutaminase antibodies, and anti-thyroid peroxidase antibodies.‡ Lowest hemoglobin level of 147 measurements.§ Lowest platelet count of 147 measurements.‖ Lowest white blood cell count of 147 measurements.¶ Normal proportions of T, B, and natural killer cells but in low numbers.# Test repeated 4 times; normal proportion of naive cells on 3 occasions (54% to 64%) and numbers of reduced naive T cells for age on 1 occasion (35%).∗∗ IgG antibodies to tetanus normal (0.04 IU/mL; range, >0.1 IU/mL); IgG antibodies to pneumococcus (all <0.35 μg/mL; range, >0.35 μg/mL).†† Very few cells available for analysis. Perforin was expressed, but quantification was not possible.‡‡ One synonymous polymorphism in PRF1, c.C900T p.H330H, identified with an Exome Aggregation Consortium frequency of 63%.§§ Test only completed on 1 occasion, with subsequent efforts hampered by insufficient cells.‖‖ TIGER (Targeted Immuno and Gastro Enrichment) panel covering 72 genes implicated in immunodeficiency and 53 genes implicated in diseases of interest to gastroenterology.¶¶ Low for age.## Highest of 2 repeats (range, 4301-4702 pg/mL).∗∗∗ Healthy children and adults have soluble CD25 levels of less than 2500 pg/mL. Children with inflammation have levels of 2500 pg/mL or greater. To date, genetically confirmed HLHs typically have had soluble CD25 levels of greater than 5000 pg/mL, but this decreases with treatment.††† Greatest value of 2 measurements (range, 450-472 mg/kg).‡‡‡ Greatest value of 43 measurements (7371 ng/mL).§§§ Test repeated 8 times, 2 times with normal results, 4 times absent, 1 time impaired, and 1 time unable to analyze. Open table in a new tab Table E2List of 18 candidate genes identified after homozygosity mapping and whole-exome sequencing (see main text)GeneVariantPredicted effect on protein from wANNOVAR summaryCommentaryANP32Cc.C592A: p.Q198KUnknownExcluded from further considerationAPC2c.C796A:p.P266TSIFT, T; Polyphen2, PExcluded from further considerationVMACc.C328T:p.R110CSIFT, T; Polyphen2, BVariant not confirmed after Sanger sequencing (ie, false positive) and therefore discountedEMR1c.A2180T:p.E727VPolyPhen2, B; present in 1% of the populationExcluded from further considerationKANK3c.G2078A:p.S693NSIFT, T; Polyphen2, D; present in 1% of the populationExcluded from further considerationMUC16c.G22123C:p.A7375PSIFT, D; Polyphen2, DConfirmed on Sanger sequencing but ultimately deemed a biologically implausible candidateZNF560c.G1253A:p.G418DSIFT, T; Polyphen2, B; present in 1% of the populationExcluded from further considerationLDLRc.G910A:p.D304NSIFT, T; Polyphen2, BBiologically implausible candidate; excluded from further considerationDNASE2c.A284G:p.Y95CSIFT, D; Polyphen2, DConfirmed on Sanger sequencing; final variant selected for further in-depth functional work (see main text)OR10C1c.A736T:p.M246LSIFT – D, Polyphen2 - BExcluded from further considerationVARS2c.G2308A:p.V770MSIFT, T; Polyphen2, DConfirmed on Sanger sequencing; variant causes mitochondrial disease (wrong phenotype) and was ultimately excluded in patient V-2 after normal respiratory chain enzyme analysis of muscle biopsy specimenMUC22c.C323G:p.T108SNo predictionExcluded from further considerationHLA-Bc.C341A:p.A114Dc.A301G:p.S101GSIFT, T; Polyphen2, BSIFT, T; Polyphen2, BBiologically implausible candidate; excluded from further considerationMICAc.G493A:p.V165ISIFT, T; Polyphen2, BConfirmed on Sanger sequencing; highly polymorphic gene, predicted nondamaging variant and therefore was excludedLY6G6Fc.G442C:p.V148LSIFT, D; Polyphen2, DConfirmed on Sanger sequencing; involved in platelet activation and therefore a biologically less plausible candidateHLA-DRB5c.C308T:p.A103Vc.G307C:p.A103PSIFT, T; Polyphen2, PSIFT, T; Polyphen2, PExcluded from further considerationHLA-DQA1c.C592A:p.Q198KSIFT, T; Polyphen2, BExcluded from further considerationFrequency (<0.01) in population was taken from the 1000 Genomes Project and the Exome Sequencing Project. Genes highlighted in boldface were considered for further study after filtering based on population frequency and biological plausibility (see main text).SIFT/PolyPhen2 predictions: D, deleterious; M, medium; P, polymorphism; T, tolerated. Open table in a new tab NK, Natural killer. Frequency (<0.01) in population was taken from the 1000 Genomes Project and the Exome Sequencing Project. Genes highlighted in boldface were considered for further study after filtering based on population frequency and biological plausibility (see main text). SIFT/PolyPhen2 predictions: D, deleterious; M, medium; P, polymorphism; T, tolerated.