Successful discontinuation of immunoglobulin G replacement at age 10 in a patient with immunoglobulin G2 deficiency.

CONTEXT: Immunoglobulin G2 deficiency that persists beyond the age of 6 years is likely to be permanent. CASE REPORT: We report on a young Japanese female, diagnosed as having immunoglobulin G2 deficiency and low anti-pneumococcal immunoglobulin G2 antibody levels when 3 years old, with a subsequent medical history of frequent respiratory infections and asthma. Monthly intravenous immunoglobulin replacement therapy was started at 4 years of age. After 8 years of age, an anti-pneumococcal immunoglobulin G2 trough level could be maintained with administration intervals longer than 6 weeks, and after 9 years and 10 months of age, therapy was discontinued. The frequency of hospital admissions was reduced by the introduction of the replacement therapy (from 8.4 times/year before the introduction to 1.1 times/year during the therapy). The patient was also able to discontinue daily medications for asthma, and serum immunoglobulin G2 was maintained at a normal level even after the cessation of replacement therapy. CONCLUSION: Termination of immunoglobulin replacement therapy in a patient with a symptomatic immunoglobulin G2 deficiency is possible, even for a child older than 6 years.

Outcomes in two Japanese adenosine deaminase-deficiency patients treated by stem cell gene therapy with no cytoreductive conditioning.

OBJECTIVE: We here describe treatment outcomes in two adenosine deaminase (ADA)-deficiency patients (pt) who received stem cell gene therapy (SCGT) with no cytoreductive conditioning. As this protocol has features distinct from those of other clinical trials, its results provide insights into SCGT for ADA deficiency. PATIENTS AND METHODS: Pt 1 was treated at age 4.7 years, whereas pt 2, who had previously received T-cell gene therapy, was treated at age 13 years. Bone marrow CD34(+) cells were harvested after enzyme replacement therapy (ERT) was withdrawn; following transduction of ADA cDNA by the γ-retroviral vector GCsapM-ADA, they were administered intravenously. No cytoreductive conditioning, at present considered critical for therapeutic benefit, was given before cell infusion. Hematological/immunological reconstitution kinetics, levels of systemic detoxification, gene-marking levels, and proviral insertion sites in hematopoietic cells were assessed. RESULTS: Treatment was well tolerated, and no serious adverse events were observed. Engraftment of gene-modified repopulating cells was evidenced by the appearance and maintenance of peripheral lymphocytes expressing functional ADA. Systemic detoxification was moderately achieved, allowing temporary discontinuation of ERT for 6 and 10 years in pt 1 and pt 2, respectively. Recovery of immunity remained partial, with lymphocyte counts in pts 1 and 2, peaked at 408/mm(3) and 1248/mm(3), approximately 2 and 5 years after SCGT. Vector integration site analyses confirmed that hematopoiesis was reconstituted with a limited number of clones, some of which were shown to have myelo-lymphoid potential. CONCLUSIONS: Outcomes in SCGT for ADA-SCID are described in the context of a unique protocol, which used neither ERT nor cytoreductive conditioning. Although proven safe, immune reconstitution was partial and temporary. Our results reiterate the importance of cytoreductive conditioning to ensure greater benefits from SCGT.

Intracellular estrogen receptor-binding fragment-associated antigen 9 exerts in vivo tumor-promoting effects via its coiled-coil region.

Estrogen receptor-binding fragment-associated antigen 9 (EBAG9) is a tumor-promoting factor of largely unknown function. To assess a causative role of EBAG9 in advanced malignancies, we generated the EG7-OVA and MethA murine tumor cell lines that stably express full-length or truncated EBAG9 protein, using retroviral-mediated gene transduction. Upon subcutaneous inoculation into immunocompetent mice, both cell lines showed marked acceleration of in vivo tumor growth when full-length EBAG9 was overexpressed. Interestingly, deletion of the coiled-coil region, thereby producing truncated EBAG9 protein, abolished the tumor-acceleration effect, establishing the importance of this domain in EBAG9-mediated tumor promotion. However, there was no alteration in in vitro cell proliferation or expression levels of MHC class I and co-stimulatory molecules believed to play a role in immune evasion of tumor cells in these tumor cell lines expressing full-length or truncated EBAG9 protein. Furthermore, both full-length and truncated EBAG9 proteins showed a predominantly cytoplasmic localization in the tumor cells. Collectively, these results suggest that EBAG9 overexpression can be causative in enhancing the malignant properties of tumor cells, and that tumor promotion likely requires EBAG9 intracellular association with as yet unidentified binding partners via the coiled-coil region.

Possible application of flow cytometry for evaluation of the structure and functional status of WASP in peripheral blood mononuclear cells.

The Wiskott-Aldrich syndrome protein (WASP), which is defective in Wiskott-Aldrich syndrome (WAS) patients, is an intracellular protein expressed in non-erythroid hematopoietic cells. Previously, we have established methods to detect intracellular WASP expression in peripheral blood mononuclear cells (PBMNCs) using flow cytometric analysis (FCM-WASP) and have revealed that WAS patients showed absent or very low level intracellular WASP expression in lymphocytes and monocytes, while a significant amount of WASP was detected in those of normal individuals. We applied these methods for diagnostic screening of WAS patients and WAS carriers, as well as to the evaluation of mixed chimera in WAS patients who had previously undergone hematopoietic stem cell transplantation. During these procedures, we have noticed that lymphocytes from normal control individuals showed dual positive peaks, while their monocytes invariably showed a single sharp WASP-positive peak. To investigate the basis of the dual positive peaks (WASP(low-bright) and WASP(high-bright)), we characterized the constituent linage lymphocytes of these two WASP-positive populations. As a result, we found each WASP(low/high) population comprised different linage PBMNCs. Furthermore, we propose that the difference between the two WASP-positive peaks did not result from any difference in WASP expression in the cells, but rather from a difference in the structural and functional status of the WASP protein in the cells. It has been shown that WASP may exist in two forms; an activated or inactivated form. Thus, the structural and functional WASP status or configuration could be evaluated by flow cytometric analysis.

Detection of a novel silent deletion, a missense mutation and a nonsense mutation in TCOF1.

BACKGROUND: Treacher Collins syndrome (TCS) is a disorder of craniofacial development, that is caused by mutations in the TCOF1 gene. TCS is inherited as an autosomal dominant trait, and haploinsufficiency of the TCOF1 gene product treacle is proposed to be etiologically involved. METHODS: Mutational analysis of the TCOF1 gene was done in 10 patients diagnosed with TCS using single-strand conformation polymorphism and direct sequencing. RESULTS: Among these 10 patients, a novel 9 bp deletion was found, together with a previously reported 2 bp deletion, a novel missense mutation and a novel nonsense mutation in three different families. Familial studies allowed judgment of whether these abnormal findings were responsible for the TCS phenotype, or not. The 9 bp deletion of three amino acids Lys-Glu-Lys (1378-1380), which was located in the nuclear localization domain of treacle, seemed not essential for the treacle function. In contrast, the novel mutation of Ala26Val is considered to affect the LisH domain, an important domain of treacle. All of the mutations thus far detected in exon 5 have resulted in frameshift, but a nonsense mutation was detected (Lys159Stop). CONCLUSION: The information obtained in the present study provides additional insights into the functional domains of treacle.

The first confirmed case with C3 deficiency caused by compound heterozygous mutations in the C3 gene; a new aspect of pathogenesis for C3 deficiency.

The complement system is an ancient cascade system that has a major role in innate and adaptive immunity. Component C3 is central to the three complement pathways. Hereditary compliment 3 (C3) deficiency characterized by severe recurrent infections and immune complex disorders is extremely rare disease. Since 1972, inherited C3 deficiency has been described in many families representing a variety of national origins; however, only 8 families of these cases have been identified their genetic defects. Interestingly, all except one (incomplete analysis) were shown to harbor homozygous C3 gene mutations. Previously we proposed a hypothesis, based on the unique process of C3 synthesis; C3 deficiency is not inherited as a simple autosomal recessive trait. Here, we report the first confirmed case with C3 deficiency caused by compound heterozygous mutations. They were a novel one base insertion (3176insT) in exon 24 which is predicted to result in a frameshift and a premature downstream stop codon (K1105X) in exon 26, and a nonsense mutation of C3303G (Y1081X) in exon 26 which was previously reported as homozygous mutations. This confirmed case suggests that our proposed hypothesis has prospects of a new aspect of pathogenesis for C3 deficiency.

Human tyrosine kinase 2 deficiency reveals its requisite roles in multiple cytokine signals involved in innate and acquired immunity.

Tyrosine kinase 2 (Tyk2) is a nonreceptor tyrosine kinase that belongs to the Janus kinase (Jak) family. Here we identified a homozygous Tyk2 mutation in a patient who had been clinically diagnosed with hyper-IgE syndrome. This patient showed unusual susceptibility to various microorganisms including virus, fungi, and mycobacteria and suffered from atopic dermatitis with elevated serum IgE. The patient's cells displayed defects in multiple cytokine signaling pathways including those for type I interferon (IFN), interleukin (IL)-6, IL-10, IL-12, and IL-23. The cytokine signals were successfully restored by transducing the intact Tyk2 gene. Thus, the Tyk2 deficiency is likely to account for the patient's complex clinical manifestations, including the phenotype of impaired T helper 1 (Th1) differentiation and accelerated Th2 differentiation. This study identifies human Tyk2 deficiency and demonstrates that Tyk2 plays obligatory roles in multiple cytokine signals involved in innate and acquired immunity of humans, which differs substantially from Tyk2 function in mice.

Analysis of the COL17A1 in non-Herlitz junctional epidermolysis bullosa and amelogenesis imperfecta.

Non-Herlitz junctional epidermolysis bullosa (nH-JEB) disease manifests with skin blistering, atrophy and tooth enamel hypoplasia. The majority of patients with nH-JEB harbor mutations in COL17A1, the gene encoding type XVII collagen. Heterozygotes with a single COL17A1 mutation, nH-JEB defect carriers, may exhibit only enamel hypoplasia. In this study, to further elucidate COL17A1 mutation phenotype/ genotype correlations, we examined two unrelated families with nH-JEB. Furthermore, we hypothesized that COL17A1 mutations might underlie or worsen the enamel hypoplasia seen in amelogenesis imperfecta (AI) patients that are characterized by defects in tooth enamel formation without other systemic manifestations. We therefore conducted COL17A1 mutational analysis in three patients from two AI families. One nH-JEB patient showed no COL17A1 expression and was a compound heterozygote for the novel premature termination codon (PTC) mutations 1285delA and Q1387X. In addition, reduced COL17A1 expression was found in a second nH-JEB patient who was homozygous for the novel PTC mutation 4335delC, the most carboxyl terminal PTC mutation thus far identified. Due to nonsense mediated mRNA decay, the position of these PTC mutations is thought not to influence the effect of COL17A1 transcript loss and hence the severity of the nH-JEB phenotype. This study is the first to suggest that type XVII collagen carboxyl PTC mutations lead to restoration of truncated polypeptide expression and to a milder clinical disease severity in nH-JEB. Conversely, we failed to detect any pathogenic COL17A1 defects in AI patients, in either exon or within the intron-exon borders of AI patients. This study furthers the understanding of mutations in COL17A1 causing nH-JEB, and clearly demonstrates that the mechanism of enamel hypoplasia differs between nH-JEB and AI diseases.

Mutational analysis of the TCOF1 gene in 11 Japanese patients with Treacher Collins Syndrome and mechanism of mutagenesis.

Treacher Collins Syndrome (TCS) (OMIM 154500) is a congenital, craniofacial disorder inherited as an autosomal dominant trait. The responsible gene for TCS, TCOF1, was mapped to 5q32-33.1 and identified in 1996. Since then, TCOF1 mutations in patients with TCS have been reported from Europe, North and South America, however, no TCS cases from an Asian country have been molecularly characterized. Here we report mutational analysis for 11 Japanese patients with TCS for the first time, and have identified TCOF1 mutations in 9 of them. The mutations detected were various, but most likely all the mutations are predicted to result in a truncated gene product, known as treacle. One mutation frequently reported was included in our cases, but no missense mutations were detected. These findings are similar to those for the previous studies for TCS in other races. We have speculated about the molecular mechanisms of the mutations in most cases. Collectively, we have defined some of the characteristic molecular features commonly observed in TCS patients, irrespective of racial difference.

[Gene therapy for adenosine deaminase deficiency].

A four year-old boy with adenosine deaminase (ADA-) deficient severe combined immunodeficiency(SCID) receiving PEG-ADA was treated under a gene therapy protocol targeting peripheral blood lymphocytes (PBLs) in 1995. After eleven infusions of autologous PBLs transduced with retroviral vector LASN encoding ADAcDNA, he exhibited increased levels of the CD8+ T lymphocytes, serum immunoglobulin, specific antibodies and delayed type hypersensitivity skin tests. Follow-up studies also provided evidence of long-term persistence and function of transduced PBLs with improvement in the immune function. However, the therapeutic effect of this gene therapy has been difficult to assess because of the concomitant treatment of PEG-ADA. Two ADA-SCID patients have been currently treated with autologous bone marrow CD34+ cells engineered with a retroviral vector GCsapM-ADA after discontinuation of PEG-ADA. The restoration of intracellular ADA enzymatic activity in lymphocytes and granulocytes resulted in correction of the systemic toxicity and liver function in the absence of PEG-ADA treatment. Both patients are at home where they are clinically well, and they do not experience adversed effect, with follow up being 12 months after CD34+ cells gene therapy.

Treacher Collins syndrome with craniosynostosis, choanal atresia, and esophageal regurgitation caused by a novel nonsense mutation in TCOF1.

Treacher Collins syndrome (TCS) is caused by mutations in TCOF1 of the nonsense, small deletion, and small insertion types, which most likely result in haploinsufficiency. We report a novel de novo nonsense mutation 2731C --> T, resulting in Arg911Stop, which truncates the protein. Our patient had the classic findings of TCS, but with documented craniosynostosis, choanal atresia, and esophageal regurgitation.

Confirming or excluding the diagnosis of Wiskott-Aldrich syndrome in children with thrombocytopenia of an unknown etiology.

Early diagnosis is an important factor in a better prognosis in patients with Wiskott-Aldrich syndrome (WAS), but it is not always easy to distinguish between WAS and immune thrombocytopenic purpura on clinical grounds. To confirm or to exclude a WAS diagnosis promptly for children with thrombocytopenia, the authors performed flow cytometric screening of Wiskott-Aldrich syndrome protein (WASP) for 10 children with thrombocytopenia of an unknown etiology. Five children were diagnosed with WAS, and the remaining 5 were diagnosed as having non-WAS causes of thrombocytopenia. There were no ambiguous results, and these were confirmed by genetic analysis. The authors conclude that screening by flow cytometry for WASP is recommended for boys with persistent thrombocytopenia of an unknown etiology.

Mixed chimera status of 12 patients with Wiskott-Aldrich syndrome (WAS) after hematopoietic stem cell transplantation: evaluation by flow cytometric analysis of intracellular WAS protein expression.

Wiskott-Aldrich syndrome (WAS) is caused by defects in the WAS protein (WASP) gene on the X chromosome. We previously reported that flow cytometric analysis of intracellular WASP expression (FCM-WASP) was useful in the diagnosis of WAS in patients and carriers. In this study, we applied FCM-WASP to evaluate the mixed chimera (MC) status of 12 WAS patients who underwent hematopoietic stem cell transplantation (HST). After HST, donor- and recipient-derived peripheral blood mononuclear cells (PBMCs) could be distinguished easily with this method, since the donor cells were WASP(bright), whereas the defective recipient cells were WASP(dim). Furthermore, with use of 2-color FCM-WASP, the MC status could be characterized by cell lineage. Six of the 12 patients with WAS were found to have MC status after HST, whereas others had complete chimera status. MC status was observed in every cell lineage examined. However, among PBMCs, recipient cells were most commonly observed in the monocyte population. Finally, to investigate the naive/memory status of donor and recipient T cells in these patients, 3-color FCM-WASP using anti-CD45RA or CD45RO was performed. We found that, in contrast to WASP(bright) T cells, most WASP(dim) T cells remained naive (CD45RA(+)/RO(-)) more than a year after HST. No imbalance in the ratio of naive to memory T cells was observed in WAS patients before HST. We conclude that FCM-WASP is a potentially useful method for clinical follow-up of WAS patients who have undergone HST. Our findings may also have important implications for the role of WASP during hematopoietic development.

The role of common gamma chain in human monocytes in vivo; evaluation from the studies of X-linked severe combined immunodeficiency (X-SCID) carriers and X-SCID patients who underwent cord blood stem cell transplantation.

Expression of common gamma chain (gammac) on monocytes was studied in five carriers of X-linked severe combined immunodeficiency (X-SCID) and two X-SCID patients who underwent cord blood stem cell transplantation (CBSCT). Flow cytometric analysis revealed that both gammac-negative and positive monocytes co-existed in X-SCID carriers, whereas no gammac-negative T, B or NK cells were observed in them. Clonal analysis and reverse transcription polymerase chain reaction studies revealed that 13.2-45.0% of monocytes from these carriers expressed the mutant gammac message. X-SCID patients who received CBSCT persistently possessed the majority of gammac-negative monocytes with a good clinical course. These results, together, may indicate that gammac is not essential for monocyte development/function in vivo.

Flow cytometry analysis of adenosine deaminase (ADA) expression: a simple and reliable tool for the assessment of ADA-deficient patients before and after gene therapy.

Clinical gene therapy trials for adenosine deaminase (ADA) deficiency have shown limited success of corrective gene transfer into autologous T lymphocytes and CD34(+) cells. In these trials, the levels of gene transduction and expression in hematopoietic cells have been assessed by DNA- or RNA-based assays and measurement of ADA enzyme activity. Although informative, these methods are rarely applied to clonal analysis. The results of these assays therefore provide best estimates of transduction efficiency and gene expression in bulk populations based on the assumption that gene transfer and expression are uniformly distributed among transduced cells. As a useful additional tool for evaluation of ADA gene expression, we have developed a flow cytometry (fluorescence-activated cell sorting, FACS) assay capable of estimating the levels of intracellular ADA on a single-cell basis. We validated this technique with T cell lines and peripheral blood mononuclear cells (PBMCs) from ADA-deficient patients that showed severely reduced levels of ADA expression (ADA-dull) by FACS and Western blot analyses. After retrovirus-mediated ADA gene transfer, these cells showed clearly distinguishable populations exhibiting ADA expression (ADA-bright), thus allowing estimation of transduction efficiency. By mixing ADA-deficient and normal cells and using enzymatic amplification, we determined that our staining procedure could detect as little as 5% ADA-bright cells. This technique, therefore, will be useful to quickly assess the expression of ADA in hematopoietic cells of severe combined immunodeficient patients and represents an important tool for the follow-up of patients treated in clinical gene transfer protocols.