key: cord-304320-1oaobtlx
authors: Lee, Pui Y.; Platt, Craig D.; Weeks, Sabrina; Grace, Rachael F.; Maher, George; Gauthier, Kasey; Devana, Sridevi; Vitali, Sally; Randolph, Adrienne G.; McDonald, Douglas R.; Geha, Raif S.; Chou, Janet
title: Immune dysregulation and Multisystem Inflammatory Syndrome in Children (MIS-C) in individuals with haploinsufficiency of SOCS1
date: 2020-08-25
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2020.07.033
sha: 
doc_id: 304320
cord_uid: 1oaobtlx

Abstract Objectives To identify the mechanisms underlying the development of infection-driven autoimmune cytopenias. Background We studied two unrelated patients with immune thrombocytopenia and autoimmune hemolytic anemia in the setting of acute infections. One patient developed Multisystem Inflammatory Syndrome in Children (MIS-C) in the setting of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Methods Whole exome sequencing was performed on both patients and the impact of the identified variants were validated by functional assays using the patients’ peripheral blood mononuclear cells (PBMCs). Results Each patient was found to have a unique heterozygous truncation variant in SOCS1. Suppressor of Cytokine Signaling 1 (SOCS1) is an essential negative regulator of type I and type II interferon (IFN) signaling. The patients’ PBMCs showed increased levels of STAT1 phosphorylation and a transcriptional signature characterized by increased expression of type I and type II interferon stimulated genes and pro-apoptotic genes. The enhanced IFN signature exhibited by the patients’ unstimulated PBMCs parallels the hyperinflammatory state associated with MIS-C, suggesting the contributions of SOCS1 in regulating the inflammatory response characteristic of MIS-C. Discussion Heterozygous loss-of-function SOCS1 mutations are associated with enhanced interferon signaling and increased immune cell activation, thereby predisposing to infection-associated autoimmune cytopenias.

Suppressor of cytokine signaling 1 (SOCS1) is an essential negative regulator of type I 80 and type II interferon signaling. Of the SOCS family members, SOCS1 binds with highest affinity 81 to the substrate binding pocket of Janus kinases (JAK) 1 and JAK2, thereby inhibiting 82 phosphorylation of Signal Transducer and Activator of Transcription (STAT) 1 and STAT2. 1 

Complete deficiency of Socs1 in mice causes perinatal lethality due to type I and type II 84 interferon (IFN)-driven inflammatory disease that can be reduced by neutralizing antibodies to 85 either type I or type II IFNs or genetic deletion of their respective receptors. 2,3 Furthermore, 86 Socs1-haploinsufficient mice exhibit features of systemic lupus erythematosus, indicating the 87 importance of bi-allelic Socs1 expression for self-tolerance. 2, 4 In a recent study detailing 88 outcomes of whole-genome sequencing for patients with primary immunodeficiency, SOCS1 89 haploinsufficiency was briefly described in two individuals with recurrent bacterial infections and 90 severe multi-systemic autoimmunity. 5 However, the immunologic sequelae of SOCS1 91 haploinsufficiency in humans remain incompletely understood.

Individuals with Evans syndrome (ES) present with immune thrombocytopenia (ITP), 93 autoimmune hemolytic anemia (AIHA), and/or immune neutropenia arising from either primary 94 or secondary causes. The relative risk of ES is significantly higher in children with monogenic 95 disorders of immunity, while ES secondary to malignancy is the most common association in 96 adults. 6 Additionally, viral and bacterial infections have been identified as triggers of ES. 6 In this 97 report, we present two unrelated children with different heterozygous loss-of-function variants in 98 SOCS1, aberrant IFN signaling, and ES. One patient developed a SARS-CoV-2 infection, 99 precipitating multisystem inflammatory syndrome of children (MIS-C), which responded well to 100 treatment with intravenous immunoglobulins (IVIG) and corticosteroids.

Patient 1 presented at five months of age with fever, otitis media, oral ulcers, and developed ITP (61 x 10 3 platelets/µL, normal 215-448 x 10 3 platelets/µL; Fig. 1A ). At two years 107 of age, in addition to ITP and immune neutropenia, he developed a warm antibody AIHA with a 108 hemoglobin of 4.4 g/dL (normal 10.5-13.0 g/dL). He was initially treated with corticosteroids and 109 subsequently transitioned to mycophenolate mofetil due to recurrence of AIHA with 110 corticosteroid tapering. In addition to immune cytopenias, he had mild CD8 + T cell lymphopenia 111 and a predominance of naïve IgD + CD27 -B cells ( has not been previously reported in the Genome Aggregation Database (gnomAD). The 117 mutation arose de novo as it was absent in both parents.

Patient 2 was diagnosed with ITP at 14 years of age when he presented with diffuse 119 petechiae and a platelet count of 7 x 10 3 cells/µL (normal 180-320 x 10 3 cells/µL; Fig. 1C ). Over 120 the course of several years, he developed a weakly positive direct antiglobulin test indicative of 121 erythrocyte autoantibodies without evidence of hemolysis. He subsequently developed 122 neutropenia and lymphopenia (Fig. 1C) . His thrombocytopenia improved with the 123 thrombopoietin receptor agonist eltrombopag and mycophenolate mofetil, but he had persistent 124 neutropenia and lymphopenia with reduced numbers of CD4 + and CD8 + T cells and a 125 predominance of naïve IgD + CD27 + B cells ( 131   1D ). This variant is also absent from the gnomAD database.

In March 2020, at the age of 17 years, Patient 2 presented with shock-like physiology 133 after having two days of fever, recurrent emesis, diarrhea, and dehydration. Upon presentation, 134 he was tachycardic and hypoxic with an oxygen saturation of 88%. Chest radiography showed 135 no evidence of acute pneumonia. Laboratory evaluation revealed metabolic acidosis with a 136 bicarbonate level of 15 mmol/L (normal 20-31 mmol/L) and increased lactate level of 10 mmol/L 137 (normal 0.5-1 mmol/L). He had a warm IgG autoimmune hemolytic anemia, CD4 + and CD8 + 138 leukopenia, and thrombocytopenia ( Table 1) . He had ongoing hemolysis, as evidenced by his 139 hemoglobin of 2.5 g/dL, an elevated lactate dehydrogenase level of 1,280 units/L (normal 100-140 210 units/L), undetectable haptoglobin, and indirect hyperbilirubinemia (indirect bilirubin 7.9 141 mg/dL; normal 0-0.8 mg/dL). His C-reactive protein was elevated at 48.9 mg/L (normal <8.0 142 mg/L), as was his procalcitonin (4.28 mg/dL, normal <0.08 mg/mL). His prothrombin time was 143 elevated at 24.9 sec (normal 12 -14.6 sec). Nasopharyngeal swab was positive for SARS-CoV-144 2 by RT-PCR. Even after receiving methylprednisolone (1 mg/kg every 6 hrs) for one day, he 145 had a markedly elevated soluble IL-2 receptor level (15,990 pg/mL) that was increased from his 146 prior level of 1,313 pg/mL (normal 45-1105 pg/mL), reflecting widespread T cell activation.

Although his AIHA was initially thought to be the most prominent feature of his SARS-CoV-2 148 infection, 7 his history of fever, involvement of the gastrointestinal, hematologic, and respiratory 149 systems, and elevated inflammatory markers were consistent with the subsequently described 

we assessed expression of ISGs in the patients' unstimulated PBMCs. Notably, Patient 2 had been treated with prednisone and mycophenolic acid for over two months at the time of this monocytes from both patients compared to healthy controls (Fig. 2C) . In contrast, the 181 expression of CD32, which is not regulated by interferon signaling, was similar between patients 182 and controls (Fig. 2C) . Consistent with these findings, qPCR of unstimulated PBMCs from both 183 patients showed increased expression of two additional ISGs, IFI44 and ISG15, compared to six 184 healthy controls (Fig. 2D) . The specificity of these observations was supported by both patients' FCGR1A  FCGR1B  FCGR1C  FI27S1  00A8S1  00A9S10  LGALS3BP   GCA  GBP1  PYGL  ANKRD22   IFI44  IFIT1  IFIT2  IFIT3  IFI6  ISG15  EPSTI1  IFIT5  RSAD2  IFI27  LY6E  CXCL9  CECR1 ANKRD22 FCGR1A S100A8 S100A9 GBP1 GCA S100A12

LGALS3BP PYGL  PGLYRP1  CXCL9  LY6E  EPSTI1  IFIT3  IFI27  IFIT2  IFI44L  IFIT1  IFI6  IFI44  ISG15  IFIT5 

Chronic obstructive pulmonary disease

RSV, respiratory syncytial virus

MGUS, monoclonal gammapathy of unknown significance

Evans syndrome in a patient with COVID-19

Simultaneous onset of COVID-19 and autoimmune haemolytic anaemia

Cold agglutinin autoimmune haemolytic anaemia associated with novel coronavirus (COVID-19)

COVID-19 infection associated with autoimmune hemolytic anemia

Autoimmune haemolytic anaemia associated with COVID-19 infection

J o u r n a l P r e -p r o o f 21 Lopez et al. 22 Zagorski et al. 23 Capes et al. 24 Lazarian et al. 25 Age ( 1 Lopez et al. 2 Zagorski et al. 3 Capes et al. 4 Lazarian et al. 5 Age (