Impaired DNASE1L3 Activity from a Novel Mutation Identified in Monogenic Systemic Lupus Erythematosus

Objectives Monogenic systemic lupus erythematosus (mSLE) is a rare, severe subtype of SLE caused by single-gene mutations that often present in childhood. Although uncommon, mSLE offers valuable insights into lupus pathogenesis and potential therapeutic targets. Over 38 genes have been implicated, including DNASE1L3, which encodes a Ca 2+ /Mg 2+ -dependent endonuclease responsible for degrading extracellular nucleic acids and clearing apoptotic debris and neutrophil extracellular traps. DNASE1L3 deficiency has been associated with dysregulation of the type I interferon (IFN-I) pathway. Here, we describe a novel DNASE1L3 variant identified in a family where the pediatric proband presented with hypocomplementemic urticarial vasculitis (HUVS) and lupus nephritis. Our objective was to define the functional and clinical implications of this DNASE1L3 variant (p.T224M). Methods Clinical gene panel sequencing (Invitae) identified the variant in the proband. Sanger sequencing confirmed homozygosity in the proband and his asymptomatic younger brother, and heterozygosity in both parents. Three-dimensional protein modeling using PyMol to predict confirmation changes. HEK293T cells overexpressing wild-type and mutant DNASE1L3 constructs were assessed for expression, secretion, and nuclease activity by Western blotting and plasmid/chromatin digestion assays. Single-cell RNA sequencing (scRNA-seq) was performed on PBMCs from both parents, the asymptomatic sibling, and 2 time points from the patient to explore downstream immune effects. Results A homozygous missense variant (c.678C>T; p.T224M) in DNASE1L3 (NM_004944.2) was identified in the affected patient. Structural modeling suggested that the substitution destabilizes the Mg 2+ -binding site for enzymatic function. Overexpression studies demonstrated that T224M DNASE1L3 is normally expressed and secreted but exhibits markedly reduced nuclease activity compared to wild-type and known pathogenic variants (R206C, W215Gfs*2). Patient sera-based validation studies are underway. Preliminary scRNA-seq analysis revealed elevated expression of IFN-stimulated genes in the patient and mild increases of T, B and NK cell populations in the asymptomatic sibling with the same homozygous variant, suggesting early immune activation and potential predisposition to IFN-I pathway dysregulation. Conclusion We have identified a novel homozygous missense mutation in DNASE1L3, encoding an endonuclease previously implicated in mSLE, in a pediatric patient with HVUS and lupus nephritis. We demonstrated that this mutation has normal extracellular secretion but impaired nuclease activity, which implies poor clearance of extracellular nuclear debris likely resulting in type 1 IFN pathway stimulation, and thus leading to autoimmune disease like SLE as well as highlighting the importance of longitudinal monitoring in at-risk family members.