Genetic testing is often pursued when evaluating patients with suspected inborn errors of immunity but interpreting variants of uncertain significance (VUS) remains challenging. Functional assays are an important avenue to characterize variant pathogenicity but are infrequently commercially available. We report a single case in which research-based functional testing was critical in determining variant pathogenicity.
A 12-year-old male with a history of recurrent childhood infections presented with recurrent episodes of bone pain in the upper arms, elbows, thoracic spine, femurs, and ankles. Medical history included oral ulcers, eczema, asthma, elevated IgE, and eosinophilia. Following an extensive work-up, he was diagnosed with chronic noninfectious osteomyelitis (CNO). Immunologic evaluation, including lymphocyte flow cytometry, immunoglobulin levels, antibody vaccine responses, and neutrophil respiratory burst, was reassuring. Exome sequencing revealed tumor necrosis factor, alpha-induced protein 3 (TNFAIP3): c.1033T>C (p.Tyr345His), suggesting possible haploinsufficiency of A20 (HA20). Other family members had milder symptoms of immune dysregulation that segregated with TNFAIP3: c.1033T>C.
To test the effect of TNFAIP3: c.1033T>C on protein function, A20-deficient HEK293T cells were transfected with wild-type versus mutant TNFAIP3, and tumor necrosis factor–induced Nuclear Factor Kappa B (NF-κB) activation (luciferase) was measured. TNFAIP3: c.1033T>C failed to suppress NF-κB activation. Hence, this variant was determined to be pathogenic. This information helped guide treatment with anakinra after bisphosphonates and tumor necrosis factor alpha inhibitors failed to control disease.
A20 is a potent inhibitor of multiple pro-inflammatory pathways. HA20, caused by loss-of-function mutations in TNFAIP3, is an early-onset immune dysregulation disease. Clinical phenotypes are heterogenous and may resemble Behcet's disease, juvenile idiopathic arthritis, inflammatory bowel disease, lupus, periodic fever syndromes, or other syndromes.
The absence of easily accessible testing for several immune pathways may result in a delay of accurately diagnosing inborn errors of immunity. This case emphasizes the importance of genetic testing when seeing patients with complex immune phenotypes. After genetic variants are identified, functional assays are often needed to accurately interpret genetic findings. Furthermore, identifying aberrant immune pathways in inborn errors of immunity is critical to the process of selecting targeted therapeutics.
