Identification of mutations in the Bruton's tyrosine kinase gene,including a novel genomic rearrangements resulting in large deletion,in Korean X-linked agammaglobulinemia patients

Mutations in the Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). We identified BTK mutations in six patients with presumed XLA from unrelated Korean families. Four out of six mutations were novel: two missense mutations (P565T, C154Y), a point mutation in a splicing donor site (IVS11+1G>A), and a large deletion (a 6.1-kb deletion including BTK exons 11–18). The large deletion, identified by long-distance PCR, revealed Alu-Alu mediated recombination extended from an Alu sequence in intron 10 to another Alu sequence in intron 18, spanning a distance of 6.1 kb. The two known mutations consisted of one missense (G462D) mutation, and a point mutation in a splicing acceptor site (IVS7−9A>G). This study suggests that large genomic rearrangements involving Alu repeats are few but an important component of the spectrum of BTK mutations.     

X-linked severe combined immunodeficiency syndrome: the first Korean case with gamma c chain gene mutation and subsequent genetic counseling

X-linked severe combined immunodeficiency (X-SCID) is a rare, life-threatening immune disorder, caused by mutations in the gamma c chain gene, which encodes an essential component of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21. A 13-month-old boy with recurrent infections who had reduced serum immunoglobulin levels and decreased numbers of CD3, CD16/56 cells was evaluated for gamma c chain gene mutation and protein expression. The patient had a C-to-T point mutation at nucleotide position 690, one of the hot spots, resulting in a single amino acid substitution of cysteine for arginine (R226C), as determined by direct sequencing and PCR-RFLP. The patient's mother was a heterozygous carrier. Percutaneous umbilical cord blood sampling was performed at the 6-month of gestation in a subsequent pregnancy. As the immunophenotype of the fetus showed an identical pattern, the pregnancy was terminated and genetic analysis of the abortus confirmed recurrence. This is the first report of the molecular diagnosis of X-SCID in Korea. Genetic analysis of the gamma c chain gene is useful for definite diagnosis and genetic counseling for X-SCID.     

Chronic persistent Epstein-Barr virus infection of natural killer cells and B cells associated with granular lymphocytes expansion

B lymphocytes and epithelial cells are the only cell types known to be infected with Epstein-Barr virus (EBV) in normal individuals. Rarely, EBV also infects other cells, including natural killer (NK) cells, almost always in the context of fatal leukaemias or lymphoproliferative disorders. We report on a 6-year-old previously healthy girl who developed fevers and liver function abnormalities for 3 months. The peripheral blood revealed an abnormal expansion of large granular lymphocytes, comprising 24% of the white blood cells. Flow cytometric analysis of the peripheral blood mononuclear cells showed an abnormal increase of CD16-positive NK cells, 62% of which were EBV-infected by in situ EBER-1 hybridization. The circulating B cells were normal in number, but 18% were infected with EBV by in situ EBER-1 hybridization. Approximately 2 years after resolution of all symptoms and continued good health, 35% of the circulating mononuclear cells were EBV-infected, indicative of persistent expansion of EBV-infected cells. We conclude that abnormal expansions of EBV-infected NK and B cells can be associated with a chronic benign course.     

A case of severe recurrent hepatitis with common variable immunodeficiency.

Severe hepatitis with an indistinct etiology manifested in a 16-year-old boy who had no particular history. The histological features of the liver and clinical course of the patient were similar to those of patients with autoimmune hepatitis characterized by interface hepatitis and severe lobular inflammation of the liver and recurrent exacerbations of hepatitis. We administered intravenous glycyrrhizin preparation daily or three times a week combined with the oral administration of ursodeoxycholic acid daily throughout the term after the initial onset of disease for the control of disease activity. The normalization of the concentration of alanine aminotransferase in serum was achieved in response to the therapy during the course. The serum concentration of immunoglobulins of the patient gradually decreased from the onset of the disease to an unacceptable level without globulin preparation during the following period of 17 months. Immunological tests revealed impairment of immunoglobulin production bythe B cell population of the patient, which led to the diagnosis of the patient as common variable immunodeficiency (CVID). The patient, with improved liver histology after 27 months from the onset of disease, benefited from the current combination therapy without severe infection through the avoidance of overimmunosuppression. CVID is defined as a heterogeneous syndrome characterized by various degrees of hypogammaglobulinemia without any specific predisposing causes, frequently associated with autoimmunity. Diagnostic criteria and therapeutic options of persistent hepatitis with CVID are to be established, as discussed in the current report.     

Large deletion of the X-linked lymphoproliferative disease gene detected by fluorescence in situ hybridization

The X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterized by an abnormal responses to infection with Epstein-Barr virus (EBV), resulting in fatal infectious mononucleosis, hypogammaglobulinemia, virus-associated hemophagocytic syndrome, and malignant lymphoma. Mutations in the gene coding for a T cell-specific SLAM-associated protein (SAP) have been recently identified in XLP patients. We report on a 1-year-old boy representing fulminant hemophagocytic syndrome. He developed high fever, lymphadenopathy, hepatosplenomegaly with liver dysfunction, and pancytopenia with marrow hemophagocytosis. EBV DNA was abnormally increased in the blood. Polymerase chain reaction failed to amplify SAP mRNA and genomic DNA products from the patient' As peripheral blood. A large deletion of the SAP gene was confirmed by fluorescence in situ hybridization (FISH). FISH analysis also disclosed that the patient's mother was a carrier. We conclude that FISH can be useful in the diagnosis of XLP with large deletions of the SAP gene and its carrier state.     

Functional analysis of JAK3 mutations in transient myeloproliferative disorder and acute megakaryoblastic leukaemia accompanying Down syndrome

JAK3 mutations have been reported in transient myeloproliferative disorder (TMD) as well as in acute megakaryoblastic leukaemia of Down syndrome (DS-AMKL). However, functional consequences of the JAK3 mutations in TMD patients remain undetermined. To further understand how JAK3 mutations are involved in the development and/or progression of leukaemia in Down syndrome, additional TMD patients and the DS-AMKL cell line MGS were screened for JAK3 mutations, and we examined whether each JAK3 mutation is an activating mutation. JAK3 mutations were not detected in 10 TMD samples that had not previously been studied. Together with our previous report we detected JAK3 mutations in one in 11 TMD patients. Furthermore, this study showed for the first time that a TMD patient-derived JAK3 mutation (JAK3(I87T)), as well as two novel JAK3 mutations (JAK3(Q501H) and JAK3(R657Q)) identified in an MGS cell line, were activating mutations. Treatment of MGS cells and Ba/F3 cells expressing the JAK3 mutants with JAK3 inhibitors significantly decreased their growth and viability. These results suggest that the JAK3 activating mutation is an early event during leukaemogenesis in Down syndrome, and they provide proof-of-principle evidence that JAK3 inhibitors would have therapeutic effects on TMD and DS-AMKL patients carrying activating JAK3 mutations.