Novel mutations in a Japanese patient with CD19 deficiency.

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by recurrent bacterial infections, hypogammaglobulinemia and low to normal numbers of circulating B cells. Mutations in the ICOS, TACI and CD19 genes have recently been identified in <10% of CVID patients. We, herein, describe two novel CD19 gene disruptions in an 8-year-old Japanese boy, who had been clinically diagnosed as having CVID at the age of 5 years. Flow-cytometric analysis demonstrated absence of CD19 and reduced CD21 expression on CD20-postive peripheral blood B cells. Mutation analysis of CD19 revealed a mutation in the splice acceptor site of intron 5 (IVS5-1G>T) of the maternal allele, resulting in skipping of exon 6, and a truncated protein product. The paternal allele was disrupted by a gross deletion encompassing at least the ATP2A1, CD19 and NFATC2IP genes. The patient had a small number of IgD(-) CD27(+) memory B cells, in which somatic mutation were detected. His B cells showed substantial proliferation upon stimulation, but reduced IgG and IgA production in vitro. These findings extend the mutation spectrum of the CD19 deficiency to four, and confirm the homogeneity of the CD19 deficiency as a unique type of CVID.

A case of body stalk anomaly arising in the second baby of a triplet pregnancy after in-vitro fertilization and embryo transfer.

We report a case of body stalk anomaly arising in the second baby of a triplet pregnancy after in-vitro fertilization and embryo transfer (IVF-ET). Body stalk anomaly or limb-body wall complex is a rare congenital anomaly with a series of similar clinical manifestations and poor prognosis. IVF-ET is an effective treatment for various types of infertility. We summarize and discuss herein the relation with the sequence of genesis for such malformations and multiple pregnancies after IVF-ET.

Survival of wild polio by a patient with XLA.

BACKGROUND: X-linked agammaglobulinemia (XLA) is a primary immunodeficiency characterized by failure of B-cell differentiation and hypogammaglobulinemia. In addition to being susceptible to bacterial infections, patients with XLA are also susceptible to enteroviruses. Systemic enterocytopathogenic human orphan virus (ECHO), coxsackie virus, and vaccine-related polio infections have caused severe morbidity and high mortality rates in XLA patients. OBJECTIVE: We report a 54-year-old male with molecularly defined XLA who survived wild poliomyelitis in childhood before the diagnosis of XLA. METHODS: At age 5, in 1951, the patient contracted wild polio, characterized by diarrhea and motor weakness. He subsequently developed recurrent sinusitis, bronchitis, and pneumonia, and at age 31 was found to be hypogammaglobulinemic and was started on immunoglobulin replacement. Laboratory evaluation at age 47 revealed an immunoglobulin G of 256 mg/dL, and B-cells (CD19) of 0.1%. Mutation analysis of Bruton's tyrosine kinase revealed a 26-basepair deletion between nucleotides 146 and 173 within the plextrin homology domain, resulting in a frameshift and premature termination. CONCLUSIONS: Resolution of wild poliovirus infection is possible in patients with XLA.

Clinical and mutational characteristics of X-linked agammaglobulinemia and its carrier identified by flow cytometric assessment combined with genetic analysis.

BACKGROUND: X-linked agammaglobulinemia (XLA), caused by mutations in Bruton's tyrosine kinase (BTK ), is the most common form of inherited antibody deficiency. We previously reported that a flow cytometric evaluation of BTK expression in monocytes could easily detect XLA as well as its carrier. OBJECTIVE: Our purpose was to perform further flow cytometric analysis in additional XLA families in Japan. METHODS: In all, 106 hypogammaglobulinemic males (from 91 families) of various ages with a lack of mature B cells (<1%) were investigated. RESULTS: Flow cytometric assessment revealed the deficient BTK expression status in 78 families (93 patients), and mutations in BTK were identified in 76 of 78 families with presumed XLA. Of the patients with normal BTK expression, 2 showed missense mutations in which the normal amount of altered BTK transcript would cause the XLA phenotype. As many as 30% of these patients with XLA were clinically or genetically recognized beyond 5 years of age. Higher concentrations (>300 mg/dL) of serum IgG were evident in the cases diagnosed among adults, seemingly preventing severe infections. Fifty-seven of 70 mothers of patients with BTK deficiency were diagnosed as obligate carriers on the basis of a bimodal BTK expression pattern. Nine of the remaining 13 mothers showing nonmosaic BTK expression had no mutations in 2 alleles; surprisingly, the other 4 mothers had the mutated alleles. CONCLUSIONS: A diagnostic approach based on flow cytometric assessment for XLA should be initially considered in genetic investigation of antibody deficiencies, regardless of the patient's age.

Bruton tyrosine kinase gene mutations in Turkish patients with presumed X-linked agammaglobulinemia.

X-linked agammglobulinemia (XLA) is a ptototypical humoral immunodeficiency caused by mutations in the gene coding for Bruton tyrosine kinase (BTK). The genetic defect in XLA impairs early B cell development resulting in marked reduction of mature B cells in the blood. Studies from different countries have demonstrated that approximately 90% of males with presumed XLA bear mutations in BTK. In this study, we report for the first time the occurrence of BTK mutations in Turkey. We performed mutational analysis of the BTK gene in 16 Turkish male patients from 13 separate families with presumed XLA based on abnormally low peripheral blood B-cell numbers (lt; 1%), hypogammaglobulinemia, and recurrent bacterial infections. We found that in nine of the 13 families (69%) a Btk mutation caused XLA. Two of the mutations were previously described, but seven novel mutations were identified: two missense (Y39C, G584R), one nonsense (Q343X), and 4 deletions (1800-1821del, 1843-1847del, 1288-1292del, 291del) resulting in frameshift and premature stop codon. By contrast, no mutations in the BTK gene were identified in the other 4 families. A consanguinity in three of these families raises the possibility that mutations in other autosomal genes which affect early B cell development may contribute to their phenotype resembling XLA.

Bruton's tyrosine kinase is present in normal platelets and its absence identifies patients with X-linked agammaglobulinaemia and carrier females.

X-linked agammaglobulinaemia (XLA) is a primary immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk) and is characterized by an arrest of B-cell development. We analysed Btk protein expression in platelets using flow cytometry and found that normal platelets express large amounts of Btk. Assessment of affected males from 45 unrelated XLA families revealed that platelets of the majority of the patients (37 out of 45 families) had decreased or absent Btk expression, and that platelets from carrier females of these families had both normal and mutated Btk expression, indicating that megakaryocytes in XLA carriers undergo random X-chromosome inactivation. These observations demonstrate that Btk is not crucial for maturation of megakaryocytes and the production of platelets. No correlation between Btk expression in platelets and clinical phenotype was observed in this study. Flow cytometric evaluation using platelets is a simple and rapid method to test Btk expression. It may be used as a screening test for XLA and for carrier detection, followed, if necessary, by more expensive mutation analyses.

Unrelated umbilical cord stem cell transplantation for X-linked immunodeficiencies.

Banked unrelated umbilical cord blood matched at 5 of 6 human leukocyte antigen loci was used to reconstitute the immune system in 2 brothers with X-linked lymphoproliferative syndrome and 1 boy with X-linked hyperimmunoglobulin-M syndrome. Pretransplant cytoreduction and posttransplant graft-versus-host prophylaxis were given. Hematopoietic engraftment and correction of the genetic defects were documented by molecular techniques. Two years after transplantation, all 3 patients have normal immune systems. These reports support the wider use of banked partially matched cord blood for transplantation in primary immunodeficiencies.

Neutrophils and mononuclear cells express vascular endothelial growth factor in acute Kawasaki disease: its possible role in progression of coronary artery lesions.

Kawasaki disease (KD) is a syndrome of systemic vasculitis of unknown etiology that is complicated by coronary artery lesions (CAL), leading occasionally to cardiac ischemic sequelae. To examine whether vascular endothelial growth factor (VEGF) is responsible for CAL in KD, we determined serum VEGF levels by ELISA and peripheral blood mononuclear cell (PBMC) and neutrophil VEGF expression by immunoblot analysis. Significantly increased levels of VEGF were demonstrated in acute KD as well as in other vasculitis syndromes (p < 0.0001). In the 10 KD patients with CAL, serum VEGF levels were maximal approximately 2 wk post-onset when CAL generally develops and were significantly higher than in 20 patients without CAL (mean, 474 and 241 pg/mL, respectively; p = 0.00015). During the same period, immunoblot analysis revealed maximal VEGF expression in PBMC, corresponding to serum VEGF levels in most patients and being particularly marked in patients with CAL (p < 0.01). Neutrophils expressed VEGF only in the early stage of acute KD and declined rapidly in the majority of KD patients regardless of the presence of CAL, showing a strikingly different expression pattern than that for PBMC. Predominant VEGF expression by PBMC was also demonstrated in patients with other vasculitis syndromes and only faintly in normal controls. The results suggest that VEGF is generated dynamically in KD, presumably reflecting its disease activity. Neutrophil-derived VEGF may play a role in regulating early vascular responses, whereas PBMC-derived VEGF may contribute to later vascular injury and remodeling.

Detection of Bruton's tyrosine kinase mutations in hypogammaglobulinaemic males registered as common variable immunodeficiency (CVID) in the Japanese Immunodeficiency Registry.

CVID is frequently diagnosed in male and female individuals with hypogammaglobulinaemia of unknown aetiology. To examine the possibility that sporadic male cases with X-linked agammaglobulinaemia (XLA), which is caused by mutations in the Bruton's tyrosine kinase (Btk) gene, might be misregistered as having CVID, we employed a flow cytometric test to identify XLA in hypogammaglobulinaemic males registered as CVID in the Japanese Immunodeficiency Registry. From 30 male cases registered as having CVID between 1992 and 1998, we selected 21 males with low or unreported peripheral B cell counts. Blood samples could be obtained from 11 patients and their mothers. Using flow cytometric analysis, the Btk-deficient status in monocytes was demonstrated in seven out of nine cases with decreased numbers of peripheral B cells. The diagnosis of XLA was confirmed in each of the seven patients by demonstration of Btk gene mutations in the patients or cellular mosaicism in the mother. This study demonstrates misregistration of XLA as CVID.

Atypical X-linked agammaglobulinemia diagnosed in three adults.

OBJECT: X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies characterized from childhood by the absence of peripheral B lymphocytes, reduced levels of serum immunoglobulins and recurrent and severe bacterial infections. These characteristics are the result of Bruton's tyrosine kinase (Btk) protein deficiency in peripheral B lymphocytes. In addition to typical XLA, several atypical cases have been recognized, who exhibited mild or even no clinical symptoms, although they were definitely deficient in Btk protein (atypical XLA). In these patients peripheral B lymphocytes and serum immunoglobulins (Igs) are detectable though at a lower level than in normal people. To clarify the discrepancies between the Btk gene mutations and the phenotypes more atypical patients should be examined. In this study we evaluated the cytoplasmic Btk protein in peripheral monocytes of some hypogammaglobulinemia adults by means of flowcytometric analysis. MATERIALS AND METHODS: Heparinized venous blood samples were collected from some hypogammaglobulinemia adults. Mononuclear cells were separated from their blood and first reacted with a phycoerythrin-labeled CD14 monoclonal antibody (MoAb) (staining of monocyte membrane). Next, the cells were fixed and permeabilized. And then these permeabilized cells were reacted with an anti-Btk MoAb (staining of cytoplasmic Btk protein) and incubated with a FITC-conjugated goat antimouse IgG1. The double-stained cells were analyzed on a flowcytometer. RESULTS AND CONCLUSION: By means of flowcytometric analysis we diagnosed three hypogammaglobulinemia adults as XLA, who did not show typical clinical progress of XLA. Advancements in diagnostic methods has facilitated a prompt and definite diagnosis of this disease.

[The oldest case with X-linked agammaglobulinemia in Japan lacking Bruton-type tyrosine kinase protein detected by flow cytometry].

We report a case of sporadic X-linked agammaglobulinemia previously diagnosed as variable immunodeficiency (VID). An 39-year-old male had recurrent episodes of respiratory tract infection since his early childhood. At the age of four, he developed partial paresis of the left limbs after polio immunization. After diagnosis of VID based on marked decrease of serum IgG, IgA and IgM levels and no antibody production against antigenetic stimuli at age 22 years old, he received intravenous immunoglobulin supplementation irregularly. We reexamined him and found marked decrease in B cells in the peripheral blood. In addition, we investigated the expression of Bruton-type tyrosine kinase on monocytes by flow cytometry and confirmed its deficiency. His mother was diagnosed as a carrier of XLA. The patient is probably the oldest case with XLA in Japan.

Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection.

The B-cell defect in X-linked agammaglobulinemia (XLA) is caused by mutations in the gene for Bruton's tyrosine kinase (BTK). Using the anti-BTK monoclonal antibody (48-2H), a flow cytometric analysis of intracytoplasmic BTK protein expressed in monocytes was successfully performed. To examine the possible identification of XLA patients and female carriers by this assay, we studied 41 unrelated XLA families with (35) or without (6) known BTK mutations. A flow cytometric assay showed deficient expression of the BTK protein in 40 of 41 patients, complete BTK deficiency in 35, and partial BTK deficiency in 5. One patient exhibited a normal level of BTK expression. All 6 patients with partial BTK deficiency or normal BTK expression had missense BTK mutations. The cellular mosaicism of BTK expression in monocytes from obligate carriers was clearly shown in 35 of 41 families. The results suggested that most BTK mutations in XLA might result in deficient expression of the BTK protein. We conclude that deficient expression of BTK protein can be evaluated by a flow cytometric assay, and the clinical usefulness and limitations in diagnosis of XLA patients and carriers are discussed.