Functional Validation of an ADA Variant in Secondary HLH Reveals Delayed Onset ADA-SCID

Introduction Hyperinflammatory and hemophagocytic lymphohistiocytosis (HLH)-like presentations are increasingly recognized as clinical “red flags” for inborn errors of immunity (IEI), including severe combined immunodeficiency (SCID). While standard newborn screening (NBS) using T cell receptor excision circles (TREC) allows for early detection of SCID, delayed presentations—particularly caused by defects in adenosine deaminase (ADA)—may be missed by NBS. Therefore, despite normal NBS, SCID should be considered in children presenting with HLH features. Case Presentation A 2-year-old male with normal NBS and a history of recurrent respiratory tract infections was admitted for fever and malaise. Laboratory evaluation demonstrated pancytopenia, elevated ferritin, hypertriglyceridemia, and hypofibrinogenemia, suggesting HLH. He subsequently developed multisystem organ failure and septicemia with Escherichia coli, Candida, adenovirus, and enterovirus. He quickly deteriorated despite aggressive therapy and passed. Genetic testing resulted postmortem and showed a heterozygous pathogenic variant in ADA, c.632G>A (p.Arg211His) and a variant of uncertain significance (VUS) in ADA, c.362+5G>C (intronic), which was not diagnostic for autosomal recessive ADA-SCID. Methods A CRISPR-engineered Jurkat T cell line carrying a homozygous patient-specific intronic splice variant was generated and compared to wild-type Jurkat controls for functional assessment of variant pathogenicity. Validation assays included: ADA enzyme activity measurement, western blot, evaluation of T cell activation/exhaustion through flow cytometry with stimulation, and bulk RNA sequencing to assess transcriptional signatures. Results ADA enzyme activity was absent in lysates from the variant Jurkat line compared with robust activity in wild-type cells. Western blot targeting the downstream epitope of the splice site revealed absent protein expression in variant cells and full-length product in wild-type controls. Compared with the wild type, the variant Jurkat line demonstrated attenuated upregulation of exhaustion marker, PD-1, despite elevated CD69 following stimulation. Bulk RNA sequencing showed upregulation of inflammatory pathways, including interferon gamma, IL-2, IL-6, and TNFα via NFκB in the variant. Discussion These findings confirm the variant’s pathogenicity, enable variant reclassification, and demonstrate a hyperinflammatory T cell response in vitro that mirrors the patient’s clinical phenotype. This integrated clinical-genetic-functional approach bridges gaps in genetic findings to actionable IEI diagnoses. Although not altering the patient’s fatal outcome, it enabled early detection and ADA-enzyme replacement therapy for his affected brother.