A 7-year-old girl from Tijuana with two healthy younger siblings from non-consanguineous parents. At age 5, she began with recurrent episodes of hepatitis, presenting with high-grade fever, abdominal pain, nausea, vomiting, diarrhea, and weight loss, associated with increased transaminases and bilirubin. Serology was negative for hepatitis virus (HBV, HCV, HAV, and HEV). During the second episode, she was found to have IgG antibodies (IgM-negative) to antiviral capsid antigen (VCA), Epstein-Barr nuclear antigen (EBNA), and early antigen. Quantification of EBV DNA by polymerase chain reaction (PCR) in plasma was persistently positive over several months (328,107 copies/ml), and she was diagnosed with chronic active EBV (CAEBV) infection.
The abdominal ultrasound was normal. Cryptosporidium and clostridium were ruled out. A hepatic biopsy showed active diffuse inflammation with lymphocytes, plasmacytes, neutrophils, and eosinophils expanding into the portal space and producing centrilobular necrosis; EBV early RNA (EBER) was positive, predominantly in CD3+CD8+ T cells (Figure 1). Colonoscopy revealed pancolitis with ulcers, and histopathology showed chronic active enterocolitis associated with EBV, with T-cell predominant EBER; quantification of EBV DNA in the gastrointestinal tract was above 7’000,000 copies per mL. EBV-associated hepatitis and enteropathy were diagnosed.
Blood counts were normal, with 1,500 peripheral lymphocytes, and 400 monocytes. Immunological workup revealed an impaired lymphoproliferation of phytohemagglutinin (PHA)-stimulated CD3+ T cells, (Figure 2A). Moreover, the patient lymphocyte subsets were shown to have decreased central (CD45RA- CCR7+) and effector (CD45RA- CCR7-) memory CD8+ T-cells, with expanded senescent CD4+ CD57+ T-cells (Figure 2B and 2C). NK cell function was normal based on degranulation assays (Figure 3A). In contrast, the expression of various cell surface markers was abnormal in NK cells from the patient as compared to healthy control, suggesting impaired NK cell differentiation (Figure 3B). All these assays were performed while the patient was receiving immunomodulatory treatment. Flow cytometry for B cell subsets and Tregs found decreased plasmablasts, memory B cells, and low Tregs (Figure 4).
Whole exome sequencing (WES) identified a novel (gnomAD allele count 0), heterozygous (MAB 0.51, DP 74x) missense variant in exon 4 (between the transcription activating TAD1 and auxiliary export AED domains) of NFAT5 (c.335C>T, p.Ser112Phe or p.Ser94Phe), likely pathogenic (SIFT 0, PolyPhen2 0.986, CADD Phred 25.8), at a position highly conserved across species (GERP++ RS 5.67). See figure 5. MutPred Top5 features predict a loss of glycosylation (p=0.007) and loss of phosphorylation at S94 (p=0.019), with a gain of a sheet (p=0.047). Family segregation through Sanger sequencing confirmed the variant to be de novo, as both parents had wild-type alleles.
She received treatment with ursodeoxycholic acid, cholestyramine, mesalazine, high-dose intravenous immunoglobulin, enteral immunoglobulin, rituximab, corticosteroids, azathioprine, and cyclosporine, with little improvement; she was later started on tocilizumab with clinical and EBV viral load improvement.
The patient underwent allogeneic hematopoietic stem cell transplantation (HSCT) from her haploidentical father, after cyclophosphamide Treg depletion and reduced-intensity conditioning; initially with control of the EBV infection (undetectable, down from 10,232cp/ml pre-HSCT). She received 4 donor lymphocyte infusions, despite which she evolved to secondary graft failure by day +220. She is currently 8 months post-transplant with mixed micro-chimerism (2.74%) and EBV viral load reactivation (1,531cp/ml), without clinical signs of disease.
A previously healthy adolescent male from Mexico City, who at 16 presented with hemophagocytic lymphohistiocytosis (HLH) associated with EBV infection. He had two healthy sisters, with no family history of consanguinity. At ages 3 and 6 years, he suffered fissured clavicle, rotula, and forearm fracture associated with traumatisms. He also had allergies to penicillin, fava beans, and some fruits. When he was 15 years old, he developed a urinary tract infection.
He started at age 16 with fever, cytopenia, triglyceridemia, and documented hemophagocytosis, for which he was treated with cyclosporin, dexamethasone, and etoposide. Soon after discharge, he was readmitted with fever, hepatosplenomegaly, oral candidiasis, and herpetic stomatitis.
Blood counts showed pancytopenia, with Hb 7.5g/dL, white blood cells 2,200 (down to 300), neutrophils 1500 (down to 200), lymphocytes 1,100 (down to 14), monocytes 100, and platelets 19,000-29,000/mm3. Ferritin 14,724-225,300 ng/ml, Serum immunoglobulins: IgG 515-1,280 mg/dL, IgM 38, IgA 272 mg/dL. IgG1 766, IgG2 154, IgG3 28.3, IgG4 16.9mg/dl. Serum autoantibodies (anti-Ro/La, ANA, lupus anticoagulant), and serologies for HIV, HBV, HCV, syphilis, and brucellosis, were all negative. EBV serum antibodies VCA (IgG), EA, and EBNA. PCR identified 2,580 copies/ml of EBV in serum, and up to 239,411 cp/ml in bone marrow. A bone marrow aspirate (BMA) was normocellular, with megaloblastic changes and active hemophagocytosis. A second BMA found hypocellularity, low megakaryocytes, and 8 hemophagocytes, with EBER diffusely positive in numerous cells.
In addition to the HLH-2004 protocol (etoposide, dexamethasone, and cyclosporine) and high-dose intravenous immunoglobulin, the patient received treatment with blood transfusions and filgrastim. He was admitted to the intensive care unit and required mechanical ventilation, despite which he progressed to disseminated intravascular coagulation with multiorgan failure and perished. Given the rapid course of the disease, further immunologic studies could not be performed.
A post-mortem WES analysis revealed a very rare heterozygous missense variant, predicted as pathogenic and highly conserved, in exon 7 of 15 of NFAT5, c.1291A>T (p.Thr431Ser, or p.Thr449Ser, p.Thr355Ser), in the DNA-binding domain RHD (Rel homology domain); gnomAD exomes allele count 1, SIFT 0.04, PolyPhen2 0.996, CADD Phred 24.6 (Minimum significance cutoff 3.3). GERP++ RS 4.85. MutPred Top5 features predict a loss of sheet (p=0.014) and gain of a loop (p=0.024). See figure 5.
A literature review on PubMed Medline searching for: (NFAT5 AND deficiency AND patient) without filters, retrieved 5 results and identified only one previously published human patient with NFAT5 haploinsufficiency (vide infra).