The fatal immune dysregulation that sometimes follows EBV infection in boys has been linked to mutations in two X chromosome-encoded genes, SLAM-associated protein (SAP) and X-linked inhibitor of apoptosis (XIAP). In this study we describe 2 girls from a consanguineous Turkish family who died after developing severe immune dysregulation and therapy-resistant EBV-positive B cell proliferation following EBV infection. SNP array-based genome-wide linkage analysis revealed IL-2-inducible T cell kinase (ITK) as a candidate gene for this immunodeficiency syndrome. Both girls harbored a homozygous missense mutation that led to substitution of a highly conserved residue (R335W) in the SH2 domain of ITK. Characteristics of ITK deficiency in mouse models, such as absence of NKT cells and high levels of eomesodermin in CD8 + cells, were seen in either one or both of the girls. Two lines of evidence suggested that R335W caused instability of the ITK protein. First, in silico modeling of the mutant protein predicted destabilization of the SH2 domain. Additionally, Western blot analysis revealed that, unlike wild-type ITK, the R335W mutant was nearly undetectable when expressed in 293 T cells. Our results suggest that ITK deficiency causes what we believe to be a novel immunodeficiency syndrome that leads to a fatal inadequate immune response to EBV. Because ITK deficiency resembles EBV-associated lymphoproliferative disorders in boys, we suggest that this molecular cause should be considered during diagnosis and treatment.

We describe three unrelated girls who had an immunodeficiency disease with granulomas in the skin, mucous membranes, and internal organs. All three girls had severe complications after viral infections, including B-cell lymphoma associated with Epstein-Barr virus (EBV). Other findings were hypogammaglobulinemia, a diminished number of T and B cells, and sparse thymic tissue on ultrasonography. Molecular analysis revealed that the patients were compound heterozygotes for mutations in recombination activating gene 1 or 2 (RAG1 or RAG2). In each case, both parents were heterozygous carriers of a RAG mutation. The mutations were associated with reduced function of RAG in vitro (3 to 30% of normal activity). The parents and one sibling in the three families were healthy.

Homozygocity mappingWe searched for homozygous regions in the DNA samples of subjects IV-4 and IV-3 using GeneChip Human Mapping 250K NspI Array of Affymetrix. The chip allows genotyping of SNPs with an average distance of approximately 50 kb between the markers. Digestion with NspI, ligation of the adaptors, and amplification with generic primers that recognize the adaptor sequence were followed by fragmentation, end labeling, and hybridization to the chip in accordance with the manufacturer's instructions. Homozygous regions greater than 5.0 Mb were manually detected. 15 Mutation analysisMutation analysis was performed by direct sequencing of PCR fragments obtained after nested amplification of the exonic and flanking intronic region coding sequences of ITK 17 exons. Primers to amplify the genomic DNA samples were designed according to GenBank sequences. Direct cycle sequencing of all PCR fragments was performed with BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and analyzed by capillary electrophoresis on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems). Analyzed sequences were compared with the cDNA and genomic DNA sequences in GenBank accession numbers NM_005546 (human ITK mRNA). Constructs and immunoblot analyses Results and Discussion Clinical phenotypeThe family tree and a clinical summary of the affected family are presented in Figure 1A and Table 1. Subject IV-5 presented at the age of 4.5 years with fever, lymphadenopathy and splenomegaly. Her past history was significant for recurrent febrile episodes which had started at four years of age. Lymph node biopsy revealed Hodgkin's lymphoma, CD30+ and EBV-LMP + and CD20-. She was treated with a regimen for advanced stage Hodgkin's disease with a good response. Four months off IL-2-inducible T-cell kinase deficiency haematologica | 2011; 96(3) 473 therapy she presented with fever, skin rash, lung disease, splenomegaly, trilineage cytopenia, hypertriglyceridemia, hypofibrinogenemia and hyperferritinemia. Bone marrow evaluation showed hemophagocytosis with Hodgkin cells positive for CD20, CD30 and EBV-LMP. Serum EBV PCR showed 125,850 copies/mL and positive EBNA IgG.Immunoglobulins were low (IgG 463 mg/dL, undetectable IgA and IgM). She was diagnosed with relapsed Hodgkin's lymphoma and hemophagocytic lymphohistiocytosis. Therapy with steroids, rituximab and chemotherapy (VP-16, vinorelbine and gemcitabine) was started but she developed further disease progression with respiratory failure and died. Subject IV-3 presented at five years of age with fever and lymphadenopathy. His past history included a profound sensorineural hearing defect, mild mental retardation and recurrent infections. Laboratory tests showed hypogammaglobulinemia (IgG 291 mg/dL, IgA and IgM below 42 and 32, respectively), positive anti-EBV VCA IgG, and negative anti-EBNA. EBV PCR was not carried out. A lymph node biopsy showed mixed cellularity Hodgkin's lymphoma with numerous Hodgkin's cells and few CD30 positive and CD20 negative Reed-Sternberg cells...