<h3>Objective:</h3> We sought to determine the etiology of <i>RANBP2</i>-negative pediatric patients presenting with acute necrotizing encephalopathy of childhood (ANEC). <h3>Background:</h3> ANEC is a rare childhood syndrome associated with fulminant encephalopathy and characteristic neuroimaging typically precipitated by acute infection and often associated with heterozygous mutation in <i>RANBP2</i>. Additional genes implicated in ANEC include <i>CPT2, MC1DN1</i> and <i>ILAE4</i>, yet many patients who meet clinical criteria for ANEC do not have mutations in any known genes and the cause of these cases has remained cryptic. <h3>Design/Methods:</h3> We performed a multicenter trio-based whole exome analysis of 12 <i>RANBP2</i>-negative pediatric patients diagnosed with ANEC using our established pipeline and filtering criteria. Whole exome sequencing was performed using a TruSeq exome capture kit and run on an Illumina NovaSeq 6000. Fastq files were analyzed using our established GATK-based pipeline and filtered for rare variants using our published approach. We prioritized rare candidate genes to determine plausible candidates based on the existing literature. <h3>Results:</h3> 32 candidate genes survived filtering and were considered in our analysis. We identified three high-confidence candidate genes. The first was <i>SCN1A</i>, segregating a heterozygous <i>de novo</i> (p.F1765Cfs*28). SCN1A encodes a voltage-gated sodium channel subunit. Mutations in SCN1A are well-known causes of Dravet Syndrome and other forms of epilepsy, but have also been associated with a fulminant encephalopathy phenotype independent of seizure burden. The second was <i>TNFAIP3</i> (heterozygous <i>de novo</i> p.R728C), previously shown to lead to an autoinflammatory Behcet-like syndrome. The third was <i>PRF1</i> (homozygous p.R225W), associated with a hemophagocytic lymphocystiocytosis-like phenotype with encephalopathy, ataxia, deep white matter lesions, and increased intracranial pressure. <h3>Conclusions:</h3> Our study indicates that ANEC is genetically heterogeneous and highlights three additional genes that should be considered in individuals presenting with ANEC. Our results also showcase how monogenic conditions can impact immune system regulation and lead to neuroimmunologic disorders. <b>Disclosure:</b> Dr. Vaidyanathan has nothing to disclose. Somayeh Bakhtiari has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Xilis Inc. Somayeh Bakhtiari has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant (Bioinformatics) with Phoenix Childrens Hospital. Dr. Appavu has received personal compensation in the range of $0-$499 for serving on a Speakers Bureau for Natus. The institution of Dr. Appavu has received research support from American Heart Association. The institution of Dr. Appavu has received research support from United States Department of Defense. Derek Neilson has nothing to disclose. The institution of Dr. Kruer has received research support from NIH NINDs. Dr. Kruer has received research support from Cure CP. Dr. Kruer has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant with NHRSA.