Background: STAT1 gain-of-function (GOF) is an immune dysregulatory disorder with poorly studied genotype-phenotype correlation, impeding prognostication and early intervention. Given previous mechanistic studies, as well as anecdotal clinical reports, we sought to systematically determine whether DNA-binding domain (DBD) mutations in STAT1 result in a different phenotype than mutations in other gene domains.Methods: Negative prognostic features previously identified by the International STAT1 GOF Study Group (invasive infections, intracranial aneurysms, and malignancy), as well as other clinical features and mortality, were compared within a cohort of 30 patients with STAT1 GOF diagnosed at our center, consisting of 9 patients with DBD mutations and 21 patients with non-DBD mutations. We subsequently reanalyzed mortality data from a large, previously-published 274-patient cohort by the International STAT1 GOF Study Group.Results: While no differences were noted with respect to malignancy or symptomatic aneurysms, invasive /opportunistic infections were substantially more common among DBD patients, as were sinopulmonary infections, bronchiectasis, enteropathy, endocrinopathies, lymphoproliferative manifestations, and recurrent fevers/HLH. DBD patients also had a lower probability of survival and younger age of mortality compared with non-DBD patients. Our re-evaluation of the published data from the International STAT1 GOF Study Group revealed a similar finding of earlier mortality among patients harboring DBD mutations. Conclusion:We report that STAT1 GOF patients with DBD mutations may be regarded as a unique subgroup, impacted more by early-onset profound combined immunodeficiency and with earlier mortality. These findings may impact clinical decision making with respect to early intervention, and in particular hematopoietic stem cell transplant considerations, in such patients.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) is an autosomal dominant immune disorder marked by wide infectious predisposition, autoimmunity, vascular disease, and malignancy. Its molecular hallmark, elevated phospho-STAT1 (pSTAT1) following interferon (IFN) stimulation, is seen consistently in all patients and may not fully account for the broad phenotypic spectrum associated with this disorder. While over 100 mutations have been implicated in STAT1 GOF, genotype–phenotype correlation remains limited, and current overexpression models may be of limited use in gene expression studies. We generated heterozygous mutants in diploid HAP1 cells using CRISPR/Cas9 base-editing, targeting the endogenous STAT1 gene. Our models recapitulated the molecular phenotype of elevated pSTAT1, and were used to characterize the expression of five IFN-stimulated genes under a number of conditions. At baseline, transcriptional polarization was evident among mutants compared with wild type, and this was maintained following prolonged serum starvation. This suggests a possible role for unphosphorylated STAT1 in the pathogenesis of STAT1 GOF. Following stimulation with IFNα or IFNγ, differential patterns of gene expression emerged among mutants, including both gain and loss of transcriptional function. This work highlights the importance of modeling heterozygous conditions, and in particular transcription factor-related disorders, in a manner which accurately reflects patient genotype and molecular signature. Furthermore, we propose a complex and multifactorial transcriptional profile associated with various STAT1 mutations, adding to global efforts in establishing STAT1 GOF genotype–phenotype correlation and enhancing our understanding of disease pathogenesis.