Five-year-old boy with no relevant family background but with personal history of epilepsy, severe psychomotor retardation, microcephaly, micrognathia, hypertelorism, low-set ears, epicanthal folds, and macroglossia ( Figure 1A ).
Admitted in the context of prolonged fever and generalized lymphadenopathies (cervical, abdominal, and mediastinal). He had no other complaints.
An extensive investigation was performed and an elevated EBV viral load (50,000 copies/ml) despite a negative EBV serology was found. Prior to receiving any immunosuppressive treatment, immunologic evaluation was performed and revealed normal serum levels of IgG, IgA and IgM, normal responses to diphteria and tetanus and a severe T and NK-cell lymphopenia, as well as B-cell lymphopenia, particularly low post-germinal class-switched memory B cells ( Table 1 ).
An excisional biopsy of a lymphadenopathy was suggestive of non-malignant polymorphic EBV (EBER+) T-cell lymphoproliferative disease. This lymphoproliferation was accompanied by infiltration of the digestive tract and he developed severe upper and lower digestive bleeding with two episodes of hemorrhagic shock, needing multiple blood transfusions. A gastric ulcer biopsy confirmed a polymorphic T infiltrate (EBER+) ( Figure 1B ) and rearrangements of T cell receptors and flow cytometry excluded monoclonality. Based on the presence of EBV-LPD in a patient with personal history of microcephaly and psychomotor retardation a presumptive diagnosis of primary immunodeficiency with inability to control EBV was made. He was treated with rituximab for B lymphocyte depletion with resolution of fever and of the gastrointestinal bleeding, which were accompanied by marked decrease in the EBV viral load (viral load 50,000> 1015 copies/mL ( Figure 1D ). Hematopoietic stem cell transplantation (HSCT) was not proposed due to the personal history of severe psychomotor retardation.
Based on the pathologic findings of EBV+ T-cell LPD, and despite the absence of documented T-cell infection in the peripheral blood, cyclophosphamide was added to rituximab when the patient eventually presented a disease relapse, with fever, increasing viral load and generalized lymphadenopathies. This led to a dramatic clinical improvement and a decrease in viral load to 20 copies/ml ( Figure 1D ). Unfortunately, in the following months, the patient had several infectious episodes (namely central line related sepsis), leading to intermittent discontinuation of the immunosuppressive treatment and subsequent relapse of the disease with massive lymphoproliferation ( Figure 1C ) and increasing viral loads, eventually leading to his death.
A next-generation sequencing panel of EBV-susceptibility related genes ( Table S1 ) led to the identification of a novel c.301G>A (p.(Ala101Thr)) homozygous ZBTB24 (NM_014797.2) mutation. This residue is highly conserved and there is a physicochemical difference between alanine and threonine ( Table S2 ). This is a very rare variant and no homozygotes have been reported (total allele frequency of around 0.017%, 48 heterozygous; 0 homozygous). Most bioinformatics analysis predicted that the patient's mutation Ala101Thr was damaging (e.g., SIFT = deleterious; MutationTaster = disease causing), with a CADD score of 17,49. This led to the diagnosis of immunodeficiency, centromeric region instability and facial anomalies syndrome (ICF) type 2. Both parents were found to harbor a heterozygotic mutation which allowed family counseling. Unfortunately, the patient died before karyotyping could be performed. We performed post-mortem Southern blot analysis for SAT2/Alpha-Satellite repeat methylation but did not find the characteristic ICF-associated repeat DNA hypomethylation. This is probably explained by the unusual nature of the mutation: a homozygous missense variant in the BTB domain.